2&#39;-Arabino-fluorooligonucleotide n3&#39;--&gt;p5&#39; phosphoramidates: their synthesis and use

ABSTRACT

Oligonucleotides with a novel sugar-phosphate backbone containing at least one 2′-arabino-fluoronucleoside and an internucleoside 3′-NH—P(—O)(OR)—O-5′ linkage, where R is a positively charged counter ion or hydrogen, and methods of synthesizing and using the inventive oligonucleotides are provided. The inventive phosphoramidate 2′-arabino-fluorooligonucleotides have a high RNA binding affinity to complementary nucleic acids and are base and acid stable.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The application claims priority from U.S. Application No.60/178,248, filed Jan. 21, 2000. For purposes of prosecution in theU.S., the priority application is hereby incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to 2′-arabino-fluorooligonucleotideN3′→P5′ phosphoramidates. More particularly, the invention is directedto 2′-arabino-fluoro monomer and oligonucleotide phosphoramidatecompositions, their use as diagnostic or therapeutic agents and methodsfor synthesizing phosphoramidate oligonucleotides containing said2′-arabino-fluorooligonucleotides.

BACKGROUND OF THE INVENTION

[0003] Nucleic acid polymer chemistry has played a crucial role in manydeveloping technologies in the pharmaceutical, diagnostic, andanalytical fields, and more particularly in the subfields of antisenseand anti-gene therapeutics, combinatorial chemistry, branched DNA signalamplification, and array-based DNA diagnostics and analysis (e.g.,Uhlmann and Peyman, Chemical Reviews, 90:543-584, 1990; Milligan et al.,J. Med. Chem. 36:1923-1937, 1993; DeMesmaeker et al., Current Opinion inStructural Biology, 5:343-355, 1995; Roush, Science, 276:1192-1193,1997; Thuong et al., Angew. Chem. Int. Ed. Engl., 32:666-690, 1993;Brenner et al., Proc. Natl. Acad. Sci., 89:5381-5383, 1992; Gold et al.,Ann. Rev. Biochem., 64:763-797, 1995; Gallop et al., J. Med. Chem.,37:1233-1258, 1994; Gordon et al., J. Med. Chem., 37:1385-1401, 1994;Gryaznov, International application No. PCT/US94/07557; Urdea et al.,U.S. Pat. No. 5,124,246; Southern et al., Genomics, 13:1008-1017, 1992;McGall et al., U.S. Pat. No. 5,412,087; Fodor et al., U.S. Pat. No.5,424,186; Pirrung et al., U.S. Pat. No. 5,405,783).

[0004] Much of this chemistry has been directed to improving the bindingstrength, specificity, and nuclease resistance of natural nucleic acidpolymers, such as DNA. Unfortunately, improvements in one property, suchas nuclease resistance, often involve trade-offs against otherproperties, such as binding strength. Examples of such trade-offsabound: peptide nucleic acids (PNAs) display good nuclease resistanceand binding strength, but have reduced cellular uptake in test cultures(e.g., Hanvey et al., Science, 258:1481-1485, 1992); phosphorothioatesdisplay good nuclease resistance and solubility, but are typicallysynthesized as P-chiral mixtures and display severalsequence-non-specific biological effects (e.g., Stein et al., Science,261 :1004-1012, 1993); methylphosphonates display good nucleaseresistance and cellular uptake, but are also typically synthesized asP-chiral mixtures and have reduce duplex stability (e.g., DeMesmaeker etal. (cited above); and so on.

[0005] Recently, a new class of oligonucleotide analog has beendeveloped having so-called N3′→P5′ phosphoramidate internucleosidelinkages which display favorable binding properties, nucleaseresistance, and solubility (Gryaznov and Letsinger, Nucleic AcidsResearch, 20:3403-3409, 1992; Chen et al., Nucleic Acids Research,23:2661-2668, 1995; Gryaznov et al., Proc. Natl. Acad. Sci.,92:5798-5802, 1995; and Gryaznov et al., J. Am. Chem. Soc.,116:3143-3144, 1994). Phosphoramidate compounds contain a 3′-amino groupat each of the 2′-deoxyfuranose nucleoside residues replacing a3′-oxygen atom. The synthesis and properties of oligonucleotide N3′→P5′phosphoramidates are also described in Gryaznov et al., U.S. Pat. Nos.5,591,607; 5,599,922; 5,726,297; and Hirschbein et al., U.S. Pat. No.5,824,793.

[0006] Oligonucleotides with various modifications of theinternucleoside linkages and 2′-position of the sugar rings have beendescribed. Among these compounds are phosphodiester (PO), andphosphorothioate (PS) oligonucleotides containing 2′-fluoro substituentsin ribo- or in arabino-configurations (Kawasaki et al., J. Med. Chem.36:831-841, 1993; Ikeda et al., Nucl. Acids Res., 26:2217-2244, 1998;Kois et al., Nucleosides Nucleotides, 12:1093-1109, 1993). Of these theoligo-2′-ribo-fluoronucleotides form the most stable complexes with DNAand RNA, whereas stability of duplexes formed by the isomericoligo-2′-arabino-fluoro nucleotides is significantly lower. The duplexstabilizing effects of 2′-ribo-fluoronucleotides was mainly attributedto their C3′-endo or N-type sugar puckering (Kawasaki et al., J. Med.Chem. 36:831-841, 1993). Unfortunately, phosphodiesteroligo-2′-ribo-fluoronucleotides are not resistant to hydrolysis bycellular nucleases, and require further modification of theinternucleoside linking groups for any in vivo applications of thesecompounds. Therefore, more stable oligonucleotide phosphorothioate(Kawasaki et al., J. Med. Chem. 36:831-841, 1993) and N3′␣P5′phosphoramidates (Schultz et al., Nucl. Acids Res., 24:2966-2973, 1996),which resist enzymatic digestion were prepared. For the former compoundsintroduction of phosphorothioate linkages resulted in reduction of theduplex stability, whereas for the latter ones synergistic stabilizingeffects of both 2′-ribo-fluoro and 3′-amino groups were observed.Additionally, oligo-2′-ribo-fluoro-N3′→P5′ phosphoramidates were lessacid labile than their 2′-deoxy N3′→P5′ phosphoramidate counterparts(Schultz et al., Nucl. Acids Res., 24:2966-2973, 1996).

[0007] The oligonucleotide N3′␣P5′ phosphoramidates form unusuallystable duplexes with complementary DNA and especially RNA strands, aswell as stable triplexes with DNA duplexes, and they are also resistantto nucleases (Chen et al., Nucleic Acids Research, 23:2661-2668, 1995;Gryaznov et al., Proc. Natl. Acad. Sci, 92:5798-5802 1995). Moreoveroligonucleotide N3′→P5′ phosphoramidates were found to be more potentantisense agents than phosphorothioate derivatives both in vitro and invivo (Skorski et al., Proc. Natl. Acad. Sci., 94:3966-3971, 1997). Atthe same time the phosphoramidates apparently have a low affinity to theintra- and extracellular proteins and increased acid liability relativeto the natural phosphodiester counterparts (Gryaznov et al., NucleicAcids Research, 24:1508-1514, 1996). These two features of theoligonucleotide phosphoramidates may potentially adversely effect theirpharmacological properties for some applications. In particular, theacid stability of an oligonucleotide is an important quality given thedesire to use oligonucleotide agents as oral therapeutics.

[0008] In order to circumvent the above described problems associatedwith presently known oligonucleotide analogs, a new class of compoundswas sought that embodies the best characteristics from botholigonueleotide phosphoramidates and 2′-ribo-fluoronucleotides. Thepresent invention describes the synthesis, properties and uses ofoligonucleotide analogues containing 2′-arabino-fluoronucleosides andinternucleoside N3′␣P5′ phosphoramidate linkages.

SUMMARY OF THE INVENTION

[0009] The compositions and methods of the present invention relate tonucleosides and to polynucleotides having contiguous nucleoside subunitsjoined by intersubunit linkages. In one aspect the present inventionrelates to 2′-arabino-fluoronucleoside and to polynucleotides comprisinga plurality of nucleoside subunits comprising at least one2′-arabino-fluoronucleoside linked to at least one additional nucleosidesubunit by a N3′→P5′ phosphoramidate inter-subunit linkage. Thepolynucleotides of the present invention preferably contain at least one2′-arabino-fluorornucleoside subunit joined by a N3′→P5′ phosphoramidateintersubunit linkage defined by the formula of 3′-[—NH—P(—O)(OR)—O—]-5′,wherein R is a positively charged counter ion, hydrogen, or lower alkyl.In a preferred embodiment of the invention, R is hydrogen. The inventivepolynucleotides can be composed such that all of the intersubunitlinkages are N3′→5′ phosphoramidates. Alternatively, the polynucleotidesof the invention contain a second class of intersubunit linkages such asphosphodiester, phosphotriester, methylphosphonate, P3′→N5′phosphoramidate, N3′→P5′ thiophosphoramidate, and phosphorothioatelinkages.

[0010] An exemplary N3′→P5′ 2′-arabino-fluoro phosphoramidateoligonucleotide has the formula:

[0011] where B is a purine or pyrimidine or an analog thereof, n is aninteger between 1 and 49, R is a positively charged counter ion,hydrogen, or lower alkyl, and R₁ is selected from the group consistingof hydroxyl, amino and hydrogen. The nucleoside subunits making up thepolynucleotides of the present invention can be selected to be in adefined sequence: such as, a sequence of bases complementary to asingle-strand nucleic acid target sequence or a sequence that will allowformation of a triplex structure between the polynucleotide and a targetduplex. The inventive oligonucleotides having 2′-arabino-fluorophosphoramidate subunits and N3′→P5′ phosphoramidate intersubunitlinkages, as described above, have superior resistance to acidhydrolysis, yet retain the same thermal stability as compared tooligonucleotides containing 2′-ribo-fluoronucleotides joined byphosphodiester linkages.

[0012] The present invention also includes a method of synthesizing anoligonucleotide containing 2′-arabino-fluoronucleosides andinternucleoside N3′→P5′ phosphoramidate linkages. In this method a first3′-amino protected nucleoside is attached to a solid phase support. Theprotected 3′ amino group is then deprotected to form a free 3′ aminogroup to which a second nucleoside is added. The free 3′ amino group ofthe first nucleoside is reacted with a 3′-protectedaminonucleoside-5′-(O-cyanoethyl-N,N-diisopropylamino)-2′-arabino-fluorophosphoramidite monomer to form an oligonucleotide internucleosideN3′→P5′ phosphoramidite linkage. The phoshoramidite linkage is thenoxidized to form a phosphoramidate internucleoside linkage.

[0013] The present invention also provides 2-arabino-fluoro monomers offormula:

[0014] where B is a purine or pyrimidine or an analog thereof; R₂ is H,lower alkyl, PO₃, or PN(R₄)₂OR₅ wherein R₄ is dialkyl, and R₅ iscyano-lower alkyl; and R₃ is hydrogen or substituted or unsubstitutedtrityl. In one representative embodiment, R₂ is PN(R₄)₂OR5 wherein R₄ isdiisopropyl, R₅ is β-cyanoethyl and R₃ is monomethoxytrityl, as shown inthe formula below:

[0015] In another embodiment the constituent B is exocyclic aminoprotected. In other embodiments of the invention, when B is guanine theN2 amino group of guanine is protected with an isobutyl group, when B is2,6-diaminopurine the exocylic amine groups are protected with aphenoxyancetyl group, and when B is cytosine the N4 amino group ofcytosine is protected with a benzoyl group.

[0016] In another embodiment of the invention, a method is provided forhybridizing a 2′-arabino-fluoro oligonucleotide N3′→P5′ phosphoramidateto a nucleic acid target. First a polynucleotide comprising a definedsequence of nucleoside subunits defined by the formula:

[0017] where B is a purine or pyrimidine or an analog thereof, n is aninteger between 1 and 49 and R₁ is selected from the group consisting ofhydroxyl, amino and hydrogen. The 2′ arabino-fluoro N3′→P5′phosphoramidate polynucleotide is then contacted with the nucleic acidtarget to allow formation of a hybridization complex between thepolynucleotide and the nucleic acid target.

[0018] The present invention also includes pharmaceutical compositionsand kits for the isolation of a target RNA that include a polynucleotidehaving at least one 2′-arabino-fluoronucleoside and phosphoramidateN3′→P5′ linkage, as described above. The inventive oligonucleotides areparticularly useful in oral therapeutic applications based onhybridization, such as, antigene and antisense applications, includingthe inhibition of telomerase enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 shows a schematic outline of the step-by-step synthesis of2-arabino-fluoronucleoside monomers, B₁ corresponds to thymine oruracil, B₂ corresponds to thymine, uracil or N⁴-benzoyl-cytosine and R1is hydrogen or methyl;

[0020]FIG. 2 shows the overall synthetic scheme used to prepare theprotected purine 2′-arabino-fluoro phosphoramidite monomers of thepresent invention. B represents a base selected from the groupconsisting of adenine (A), guanine (G), and 2,6-diaminopurine (D),uracil (U), cytosine (C) and thymidine (T). Lower case letters a-dassociated with compound numbers represent the bases adenine (a),guanine (g), 2,6-diaminopurine (d), uracil (u) and thymine (t). Tol istouoyl, MMTNH is (monomethoxytrityl)amino, iPr₂N is diisopropylamino,and CEO is β-cyanoethyl, R is anisoyl when the base is G or D, andtoluoyl when the base is A, T, or U. In addition, when B is adenine theN6 amino group of adenine is protected with a benzoyl group, when B is2,6-diaminopurine the exocylic amine groups are protected with aphenoxyacetyl group, or when B is guanine the N² amino group of guanineis protected with an isobutyl group; and

[0021]FIG. 3 shows the internucleoside linkage structure of anoligonucleotide containing 2′-arabino-fluoronucleosides joined aninternucleoside N3′→P5′ phosphoramidate linkage, where R is a positivelycharged counter ion or hydrogen;

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0022] Definitions

[0023] An “alkyl group” refers to an alkyl or substituted alkyl grouphaving 1 to 20 carbon atoms, such as methyl, ethyl, propyl, and thelike. Lower alkyl typically refers to C₁ to C₅. Intermediate alkyltypically refers to C₆ to C₁₀.

[0024] An “aryl group” refers to an aromatic ring group having 5-20carbon atoms, such as phenyl, naphthyl, anthryl, or substituted arylgroups, such as alkyl- or aryl-substitutions like tolyl, ethylphenyl,biphenylyl, etc. Also included are heterocyclic aromatic ring groupshaving one or more nitrogen, oxygen, or sulfur atoms in the ring.

[0025] A “positively charged counter ion” refers to any ion capable offorming an ion pair with oxygen, such a Na⁺, K⁺, Ca⁺, Mg²⁺, Mn²⁺ and thelike.

[0026] “Oligonucleotides” typically refer to nucleoside subunit polymershaving between about 2 and about 50 contiguous subunits. The nucleosidesubunits can be joined by a variety of intersubunit linkages, including,but not limited to, those shown in FIGS. 1A to 1C. Further,“oligonucleotides” includes modifications, known to one skilled in theart, to the sugar backbone (e.g., ribose or deoxyribose subunits), thesugar (e.g., 2′ substitutions), the base, and the 3′ and 5′ termini. Theterm “polynucleotide”, as used herein, has the same meaning as“oligonucleotide” is used interchangeably with “polynucleotide”.

[0027] Whenever an oligonucleotide is represented by a sequence ofletters, such as “ATGUCCTG,” it will be understood that the nucleotidesare in 5′→3′ order from left to right and that “A” denotesdeoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine,“T” denotes thymidine, and “U” denotes deoxyuridine, unless otherwisenoted.

[0028] As used herein, “nucleoside” includes the natural nucleosides,including 2′-deoxy and 2′-hydroxyl forms, e.g., as described in Kornbergand Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).“Analogs” in reference to nucleosides includes synthetic nucleosideshaving modified base moieties and/or modified sugar moieties, e.g.,described generally by Scheit, Nucleotide Analogs (John Wiley, New York,1980). Such analogs include synthetic nucleosides designed to enhancebinding properties, e.g., stability, specificity, or the like, such asdisclosed by Uhlnann and Peyman (Chemical Reviews, 90:543-584, 1990).

[0029] A “base” is defined herein to include (i) typical DNA and RNAbases (uracil, thymine, adenine, guanine, and cytosine), and (ii)modified bases or base analogs (e.g., 5-methyl-cytosine, 5-bromouracil,or inosine). A base analog is a chemical whose molecular structuremimics that of a typical DNA or RNA base.

[0030] As used herein, “pyrimidine” means the pyrimidines occurring innatural nucleosides, including cytosine, thymine, and uracil, and commonanalogs thereof, such as those containing oxy, methyl, propynyl,methoxy, hydroxyl, amino, thio, halo, and like substituents. The term asused herein further includes pyrimidines with common protection groupsattached, such as N₄-benzoylcytosine. Further common pyrimidineprotection groups are disclosed by Beaucage and Iyer (Tetrahedron48:223-2311, 1992).

[0031] As used herein, “purine” means the purines occurring in naturalnucleosides, including adenine, guanine, and hypoxanthine, and commonanalogs thereof, such as those containing oxy, methyl, propynyl,methoxy, hydroxyl, amino, thio, halo, and like substituents. The term asused herein further includes purines with common protection groupsattached, such as N₂-benzoylguanine, N₂-isobutyrylguanine,N₆-benzoyladenine, and the like. Further common purine protection groupsare disclosed by Beaucage and Iyer (cited above).

[0032] As used herein, the term “protected” as a component of a chemicalname refers to art-recognized protection groups for a particular moietyof a compound, e.g., “5′-protected-hydroxyl” in reference to anucleoside includes triphenylmethyl (i.e., trityl),p-anisyldiphenylmethyl (i.e., monomethoxytrityl or MMT),di-p-anisylphenylmethyl (i.e., dimethoxytrityl or DMT), and the like.Art-recognized protection groups are described in the followingreferences: Gait, editor, Oligonucleotide Synthesis: A PracticalApproach (IRL Press, Oxford, 1984); Amarnath and Broom, ChemicalReviews, 77:183-217, 1977; Pon et al., Biotechniques, 6:768-775, 1988;Ohtsuka et al., Nucleic Acids Research, 10:6553-6570, 1982; Eckstein,editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press,Oxford, 1991), Greene and Wuts, Protective Groups in Organic Synthesis,Second Edition (John Wiley & Sons, New York, 1991), Narang, editor,Synthesis and Applications of DNA and RNA (Academic Press, New York,1987), Beaucage and Iyer (cited above), and like references.

[0033] As used herein, “stringency” refers to the hybridizationconditions under which an oligonucleotide binds to a nucleic acids towhich it has sequence homology, i.e. a “target nucleic acid.” It isunderstood that an oligonucleotide need not be 100% complementary to itstarget nucleic acid sequence to be specifically hybridizable. Anoligonucleotide is specifically hybridizable when binding ofoligonucleotide to the target interferes with the normal function of thetarget molecule to cause a loss of utility, and there is a sufficientdegree of complementarity to avoid nonspecific binding ofoligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment or, in the case of invitro assays, under conditions in which the assays are conducted.

[0034] The term “hybridization stringency” is well known in the art andrelates to the approximate buffer, salt and temperature conditions underwhich an oligonucleotide hybridizes specifically to its target nucleicacid. Generally, the following conditions are used to definehybridization stringency: “high stringency” denotes the use of ahybridization or wash solution comprising 10 mM phosphate buffer, pH7.0, at a range of about 45-55° C. The term “moderate stringency” meansuse of 10 mM phosphate buffer, pH 7.0, with a salt concentration ofabout 0.1 to 0.5 M NaCl, at a temperature of between about 30 to 45° C.The term “low stringency” means use of about 10 mM phosphate buffer atabout pH 7.0, 1.0 M NaCl at room temperature. Low stringency buffers mayalso include 10 mM MgCl₂. It is well understood in the art of nucleicacid hybridization that many factors, such as temperature, salt andinclusion of other components such as formamide, affect the stringencyof hybridization.

[0035] The compounds of the present invention may be used to inhibit orreduce enzyme activity, such as reducing the activity of the telomeraseenzyme and/or reducing the proliferation of cells having telomeraseactivity. In these contexts, inhibition or reduction of the enzymeactivity or cell proliferation refer to a lower level of the measuredactivity relative to a control experiment in which the enzyme or cellsare not treated with the test compound. In particular embodiments, theinhibition or reduction in the measured activity is at least a 10%reduction or inhibition. One of skill in the art will appreciate thatreduction or inhibition of the measured activity of at least 20%, 50%,75%, 90% or 100% may be preferred for particular applications.

[0036] The present invention is directed generally to2′-arabino-fluorooligonucleosides and to oligonucleotides containing atleast one 2′-arabino-fluoronucleoside joined by an internucleosideN3′→P5′ phosphoramidate linkage, methods of synthesizing such analogpolynucleotides, and methods of using the inventive oligonucleotides astherapeutic compounds and in diagnostics. More particularly, the2′-arabino-fluorooligonucleotides of the present invention have theformula:

[0037] wherein B is a purine or pyrimidine or an analog thereof such asuracil, thymine, adenine, guanine, cytosine, 5-methylcytosine,5-bromouracil and inosine,

[0038] R is a positively charged counter ion or hydrogen,

[0039] R₁ is selected from the group consisting of hydroxyl, amino andhydrogen, and

[0040] n is an integer between 1 and 49.

[0041] The nucleoside subunits making up the polynucleotides nucleotidesof the present invention can be selected to be in a defined sequence:such as, a sequence of bases complementary to a single-strand nucleicacid target sequence or a sequence that will allow formation of atriplex structure between the polynucleotide and a target duplex.

[0042] The inventive oligonucleotides can be used to hybridize to targetnucleic acid sequences such as RNA and DNA. When desirable, theoligonucleotides of the present invention can be labeled with a reportergroup such as radioactive labels, biotin labels, fluorescent labels andthe like, to facilitate the detection of the polynucleotide itself andits presence in, for example, hybridization complexes.

[0043] In another aspect of the invention, a kit for isolating a targetnucleic acid from a sample is provided. The kit contains anoligonucleotide having a defined sequence of nucleoside subunits joinedby a least one intersubunit linkage defined by the formula:

[0044] where B is a purine or pyrimidine or an analog thereof, n is aninteger between 1 and 49, R is a positively charged counter ion orhydrogen, and R₁ is selected from the group consisting of hydroxyl,amino and hydrogen, and wherein the oligonucleotide hybridizes to thetarget nucleic acid.

[0045] In other aspects, the invention is directed to a solid phasemethod of synthesizing oligonucleotide containing2′-arabino-fluoronucleosides and internucleoside N3′→P5′ phosphoramidatelinkages using a modification of the phosphoramidite transfermethodology of Nelson et al. (J. Organic Chemistry 62:7278-7287, 1997).The synthetic strategy employed 3′-NH-trityl-protected 3!aminonucleoside5′-O-cyanoethyl-N,N-diisopropylaminophosphoramidites (Nelson et al.,cited above) that were purchased from Cruachem and JBL Scientific, Inc.(Aston, Pa. and San Luis Obispo, Calif., respectively). Every syntheticcycle was conducted using the following chemical steps: 1)detritylation, 2) coupling; 3) capping, and 4) oxidizing.

[0046] Chimeric 2′-arabino-fluoro N3′→P5′-thiophosphoramidateoligonucleotides can be made by substitution of a sulfurization reactionin place of the oxidation reaction at synthetic step 4 above, whichresults in formation of a thio-phosphoramidate mixed oligonucleotide(see Pongracz, et al., Tetrahedron, 40:7661-7664, 1999). In addition,chimeric oligonucleotides can be made comprising 2′-ribo-fluoro (seeSchultz, et al., Nucl. Acids Res., 24:2966-2973, 1996) and2′-arabino-fluoro phosphoramidates, and thio-N3′→P5′-phosporamidateswith 2′-arabino-fluoronucleosides. Similarly,phosphodiester-2′-arabino-fluoro phosphoramidates can be made by using5′-phosphoramidite-3′-O-DMTr-protected nucleotides as monomeric buildingblocks. These synthetic approaches are known in the art (see Pongracz,et al., 1999 and Schultz, et al., 1996.).

[0047] In another embodiment of the present invention, the acidstability of oligonucleotides is increased by incorporating2′-arabino-fluoronucleosides subunits linked by N3′→P5′thiophosphoramidate intersubunit linkages into an oligonucleotide. Thehybridization properties of the phosphoramidate2′-arabino-fluorooligonucleotides were evaluated relative tocomplementary DNA or RNA strands having phosphodiester or2′-arabino-ribose phosphoramidate intersubunit linkages. The thermalstability data for duplexes generated from 2′-arabino-ribosephosphoramidate, phosphoramidate, phosphodiester and the inventive2′-arabino-fluoro phosphoramidate oligonucleotides, are summarized inExample 4, Table 1.

Applications of Oligonucleotides Containing 2-Arabino-Fluoronucleosidesand Internucleoside 3′-NHP(O)(O⁻)O-5′ Phosphoramidate Linkages

[0048] 2′-Arabino-fluorooligonucleotide SEQ ID NO:8 phosphoramidate wassynthesized (see Table 1). This compound was surprisingly base and acidstable and formed a stable complex with a complementary RNA and DNAtarget. The N3′→P5′ phosphoramidate 2′-arabino-fluoro-polynucleotides ofthe present invention are useful for anti-sense and anti-genediagnostic/therapeutic applications. In a preferred embodiment of thepresent invention, the oligonucleotides are oligodeoxyribonucleotides.

[0049] A. Telomerase Inhibition Applications

[0050] Recently, an understanding of the mechanisms by which normalcells reach the state of senescence, i.e., the loss of proliferativecapacity that cells normally undergo in the cellular aging process, hasbegun to emerge. The DNA at the ends, or telomeres, of the chromosomesof eukaryotes usually consists of tandemly repeated simple sequences.Scientists have long known that telomeres have an important biologicalrole in maintaining chromosome structure and function. More recently,scientists have speculated that the cumulative loss of telomeric DNAover repeated cell divisions may act as a trigger of cellular senescenceand aging, and that the regulation of telomerase, an enzyme involved inthe maintenance of telomere length, may have important biologicalimplications. See Harley, Mutation Research, 256:271-282, 1991.Experiments by Bodnar et al. have confirmed the importance of telomeresand telomerase in controlling the replicative lifespan of culturednormal human cells. See Bodnar et al., Science 279:349-352, 1998.

[0051] Telomerase is a ribonucleoprotein enzyme that synthesizes onestrand of the telomeric DNA using as a template a sequence containedwithin the RNA component of the enzyme. See Blackburn, Annu. Rev.Biochem., 61:113-129, 1992. The RNA component of human telomerase hasbeen sequenced and is 453 nucleotides in length containing a series of11-base sequence repeats that is complementary to the telomere repeat.Human telomerase activity has been inhibited by a variety ofoligonucleotides complementary to the RNA component of telomerase. SeeNorton et al., Nature Biotechnology, 14:615, 1996; Pitts et al., Proc.Natl. Acad. Sci., 95:11549-11554, 1998; and Glukhov et al., Bioch.Biophys. Res. Commun., 248:368-371, 1999. 2-Arabino-fluorophosphoramidate oligonucleotides of the present invention arecomplementary to 10 to 50 nucleotides of telomerase RNA. Preferably, theinventive telomerase inhibitor 2-arabino-fluoro phosphoramidateoligonucleotides have a 10 to 20 consecutive base sequence that arecomplementary to telomerase RNA.

[0052] Methods for detecting telomerase activity, as well as foridentifying compounds that regulate or affect telomerase activity,together with methods for therapy and diagnosis of cellular senescenceand immortalization by controlling telomere length and telomeraseactivity, have also been described. See, Feng et al., Science,269:1236-1241, 1995; Kim et al., Science, 266:2011-2014, 1994; PCTpatent publication No. 93/23572, published Nov. 25, 1993; and U.S. Pat.Nos. 5,656,638, 5,760,062, 5,767,278, 5,770,613 and 5,863,936.

[0053] The identification of compounds that inhibit telomerase activityprovides important benefits to efforts at treating human disease.Compounds that inhibit telomerase activity can be used to treattelomerase-mediated disorders, such as cancer, since cancer cellsexpress telomerase activity and normal human somatic cells do notpossess telomerase activity at biologically relevant levels (i.e., atlevels sufficient to maintain telomere length over many cell divisions).Unfortunately, few such compounds, especially compounds with highpotency or activity and compounds that are orally bioavailable, havebeen identified and characterized. Hence, there remains a need forcompounds that act as telomerase inhibitors that have relatively highpotency or activity and that are orally bioavailable, and forcompositions and methods for treating cancer and other diseases in whichtelomerase activity is present abnormally.

[0054] The new phosphoramidate 2′-arabino-fluorooligonucleotidecompounds of the present invention are acid and base stable, andtherefore, have many valuable uses as inhibitors of deleterioustelomerase activity, such as, for example, in the treatment of cancer inhumans. Pharmaceutical compositions of phosphoramidate2′-arabino-fluorooligonucleotide can be employed in treatment regimensin which cancer cells are killed, in vivo, or can be used to kill cancercells ex vivo. Thus, this invention provides therapeutic compounds andcompositions for treating cancer, and methods for treating cancer inmammals (e.g., cows, horses, sheep, steer, pigs and animals ofveterinary interest such as cats and dogs). In addition, thephosphoramidate 2′-arabino-fluorooligonucleotides of the presentinvention may also be used to treat other telomerase-mediated conditionsor diseases, such as, for example, other hyperproliferative orautoimmune disorders such as psoriasis, rheumatoid arthritis, immunesystem disorders requiring immune system suppression, immune systemreactions to poison ivy or poison oak, and the like.

[0055] As noted above, the immortalization of cells involves inter aliathe activation of telomerase. More specifically, the connection betweentelomerase activity and the ability of many tumor cell lines, includingskin, connective tissue, adipose, breast, lung, stomach, pancreas,ovary, cervix, uterus, kidney, bladder, colon, prostate, central nervoussystem (CNS), retina and blood tumor cell lines, to remain immortal hasbeen demonstrated by analysis of telomerase activity (Kim et al.,above). This analysis, supplemented by data that indicates that theshortening of telomere length can provide the signal for replicativesenescence in normal cells, see PCT Application No. 93/23572,demonstrates that inhibition of telomerase activity can be an effectiveanti-cancer therapy. Thus, telomerase activity can prevent the onset ofotherwise normal replicative senescence by preventing the normalreduction of telomere length and the concurrent cessation of cellreplication that occurs in normal somatic cells after many celldivisions. In cancer cells, where the malignant phenotype is due to lossof cell cycle or growth controls or other genetic damage, an absence oftelomerase activity permits the loss of telomeric DNA during celldivision, resulting in chromosomal rearrangements and aberrations thatlead ultimately to cell death. However, in cancer cells havingtelomerase activity, telomeric DNA is not lost during cell division,thereby allowing the cancer cells to become immortal, leading to aterminal prognosis for the patient. Agents capable of inhibitingtelomerase activity in tumor cells offer therapeutic benefits withrespect to a wide variety of cancers and other conditions (e.g., fungalinfections) in which immortalized cells having telomerase activity are afactor in disease progression or in which inhibition of telomeraseactivity is desired for treatment purposes. The telomerase inhibitors ofthe invention can also be used to inhibit telomerase activity in germline cells, which may be useful for contraceptive purposes.

[0056] In addition, it will be appreciated that therapeutic benefits fortreatment of cancer can be realized by combining a telomerase inhibitorof the invention with other anti-cancer agents, including otherinhibitors of telomerase such as described in U.S. Pat. Nos. 5,656,638,5,760,062, 5,767,278, 5,770,613 and 5,863,936. The choice of suchcombinations will depend on various factors including, but not limitedto, the type of disease, the age and general health of the patient, theaggressiveness of disease progression, the TRF length and telomeraseactivity of the diseased cells to be treated and the ability of thepatient to tolerate the agents that comprise the combination. Forexample, in cases where tumor progression has reached an advanced state,it may be advisable to combine a telomerase inhibiting compound of theinvention with other agents and therapeutic regimens that are effectiveat reducing tumor size (e.g., radiation, surgery, chemotherapy and/orhormonal treatments). In addition, in some cases it may be advisable tocombine a telomerase inhibiting agent of the invention with one or moreagents that treat the side effects of a disease, e.g., an analgesic, oragents effective to stimulate the patient's own immune response (e.g.,colony stimulating factor).

[0057] The compounds of the present invention demonstrate inhibitoryactivity against telomerase activity in vivo, as can be demonstrated asdescribed below. The in vitro activities of the compounds of theinvention can also be demonstrated using the methods described herein.As used herein, the term “in vitro” refers to tests performed usingliving cells in tissue culture. Such procedures are also known as “exvivo”.

[0058] One method used to identify phosphoramidate 2′-arabino-fluoropolynucleotides of the invention that inhibit telomerase activityinvolves placing cells, tissues, or preferably a cellular extract orother preparation containing telomerase in contact with several knownconcentrations of a phosphoramidate 2′-arabino-fluorooligonucleotidethat is complementary to the RNA component of telomerase in a buffercompatible with telomerase activity. The level of telomerase activityfor each concentration of the phosphoramidate 2′-arabino-fluoropolynucleotide is measured and the IC₅₀ (the concentration of thepolynucleotide at which the observed activity for a sample preparationis observed to fall one-half of its original or a control value) for thepolynucleotide is determined using standard techniques. Other methodsfor determining the inhibitory concentration of a compound of theinvention against telomerase can be employed as will be apparent tothose of skill in the art based on the disclosure herein.

[0059] With respect to the treatment of malignant diseases usingphosphoramidate 2′-arabino-fluoro polynucleotides that are complementaryto the RNA component of telomerase are expected to induce crisis intelomerase-positive cell lines. Treatment of telomerase-positive celllines, such as HEK-293, HeLa and HME50-5E human breast epithelial cellsthat were spontaneously immortalized, with a phosphoramidate′-arabino-fluorooligonucleotide that is complementary to the RNAsequence component of telomerase is also expected to induce a reductionof telomere length in the treated cells.

[0060] Phosphoramidate 2′-arabino-fluorooligonucleotides of theinvention are also expected to induce telomere reduction during celldivision in human tumor cell lines, such as the ovarian tumor cell linesOVCAR-5 and SK-OV-3. Importantly, however, in normal human cells used asa control, such as BJ cells of fibroblast origin, the observed reductionin telomere length is expected to be no different from cells treatedwith a control substance, e.g., a thiophosphoramidate oligonucleotidethat has at least one single base mismatch with the complementarytelomerase RNA target. The phosphoramidate2′-arabino-fluorooligonucleotides of the invention also are expected todemonstrate no significant cytotoxic effects at concentrations belowabout 20 μM in the normal cells.

[0061] In addition, the specificity of the phosphoramidate2′-arabino-fluorooligonucleotides of the present invention fortelomerase RNA can be determined by performing hybridization tests withand comparing their activity (IC₅₀) with respect to telomerase and toother enzymes known to have essential RNA components. Compounds havinglower IC₅₀ values for telomerase as compared to the IC₅₀ values towardthe other enzymes being screened are said to possess specificity fortelomerase.

[0062] In vivo testing can also be performed using a mouse xenograftmodel, for example, in which OVCAR-5 tumor cells are grafted onto nudemice, in which mice treated with a phosphoramidate2′-arabino-fluorooligonucleotide of the invention are expected to havetumor masses that, on average, may increase for a period following theinitial dosing, but will begin to shrink in mass with continuingtreatment. In contrast, mice treated with a control (e.g., aphosphoramidate 2′-arabino-fluorooligonucleotide that has at least onesingle base mismatch with the complementary telomerase RNA target) areexpected to have tumor masses that continue to increase.

[0063] From the foregoing those skilled in the art will appreciate thatthe present invention also provides methods for selecting treatmentregimens involving administration of a phosphoramidate2′-arabino-fluorooligonucleotide of the invention. For such purposes, itmay be helpful to perform a terminal restriction fragment (TRF) analysisin which DNA from tumor cells is analyzed by digestion with restrictionenzymes specific for sequences other than the telomeric (T₂ AG₃)_(N)sequence. Following digestion of the DNA, gel electrophoresis isperformed to separate the restriction fragments according to size. Theseparated fragments are then probed with nucleic acid probes specificfor telomeric sequences to determine the lengths of the terminalfragments containing the telomere DNA of the cells in the sample. Bymeasuring the length of telomeric DNA, one can estimate how long atelomerase inhibitor should be administered and whether other methods oftherapy (e.g., surgery, chemotherapy and/or radiation) should also beemployed. In addition, during treatment, one can test cells to determinewhether a decrease in telomere length over progressive cell divisions isoccurring to demonstrate treatment efficacy.

[0064] Thus, in one aspect, the present invention provides compoundsthat can serve in the war against cancer as important weapons againstmany types of malignancies. In particular, the phosphoramidate2′-arabino-fluoro-polynucleotides of the present invention can provide ahighly general method of treating many, if not most, malignancies, asdemonstrated by the highly varied human tumor cell lines and tumorshaving telomerase activity. More importantly, the phosphoramidate2′-arabino-fluorooligonucleotides of the present invention can beeffective in providing treatments that discriminate between malignantand normal cells to a high degree, avoiding many of the deleteriousside-effects present with most current chemotherapeutic regimes whichrely on agents that kill dividing cells indiscriminately.

[0065] B. Antisense Applications

[0066] Antisense therapy involves the administration of exogenousoligonucleotides that bind to a target nucleic acid, typically an RNAmolecule, located within cells. The term antisense is so given becausethe oligonucleotides are typically complementary to mRNA molecules(“sense strands”) which encode a cellular product.

[0067] The phosphoramidate 2′-arabino-fluorooligonucleotides describedherein are useful for antisense inhibition of gene expression (Matsukuraet al., Proc. Natl. Acad. Sci., 86:4244-4248, 1989; Agrawal et al.,Proc. Natl. Acad. Sci., 86:7790-7794, 1989; Zamecnik et al., Proc. Natl.Acad. Sci., 83:4143-4146, 1986; Rittner and Sczakiel, Nucleic AcidsResearch, 19:1421-1426, 1991; Stein and Cheng, Science, 261:1004-1012,1993). N3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotides havetherapeutic applications for a large number of medically significanttargets, including, but not limited to inhibition of cancer cellproliferation and interference with infectious viruses. The N3′→P5′phosphoramidate 2′-arabino-fluorooligonucleotides are useful for bothveterinary and human applications. The high acid stability of theinventive oligonucleotides and their ability to act effectively asantisense molecules at low concentrations (see below) make theseoligonucleotides highly desirable as therapeutic antisense agents.

[0068] Anti-sense agents typically need to continuously bind all targetRNA molecules so as to inactivate them or alternatively provide asubstrate for endogenous ribonuclease H (RNase H) activity. Sensitivityof RNA/oligonucleotide complexes, generated by the methods of thepresent invention, to RNase H digestion can be evaluated by standardmethods (Donia et al., J. Biol. Chem., 268:14514-14522, 1993; Kawasakiet al., J. Medicinal Chem., 36:831-841, 1993).

[0069] The methods of the present invention provide several advantagesover the more conventional antisense agents. First, phosphoramidate2′-arabino-fluorooligonucleotides bind more strongly to RNA targets ascorresponding phosphodiester oligonucleotides. Second, thephosphoramidate 2′-arabino-fluorooligonucleotides are more resistant todegradation by cellular nucleases and by acid or base conditions.

[0070] Further, when an RNA is coded by a mostly purine strand of aduplex target sequence, 2′-arabino-fluoro phosphoramidate analogoligonucleotides targeted to the duplex also have potential forinactivating the DNA—i.e., the ability to inactivate a pathogen in bothsingle-stranded and double-stranded forms (see discussion of anti-genetherapies below). Lastly, the oligonucleotides of the present inventionform more stable triple stranded structures when annealed to doublestranded DNA targets than do natural phosphodiester linkedoligonucleotides.

[0071] Sequence-specific phosphoramidate2′-arabino-fluorooligonucleotide molecules are potentially powerfultherapeutics for essentially any disease or condition that in some wayinvolves RNA. Exemplary modes by which such sequences can be targetedfor therapeutic applications include:

[0072] a) targeting RNA sequences expressing products involved in thepropagation and/or maintenance infectious agents, such as, bacteria,viruses, yeast and other fungi, for example, a specific mRNA encoded byan infectious agent;

[0073] b) formation of a duplex molecule that results in inducing thecleavage of the RNA (e.g., RNase H cleavage of RNA/DNA hybrid duplexmolecules). This is an important property of the inventive2′-arabino-fluoro phosphoramidate oligonucleotides because, in general,phosphoramidates are not good substrates for RNase H;

[0074] c) blocking the interaction of a protein with an RNA sequence(e.g., the interaction of TAT and TAR, see below); and

[0075] d) targeting sequences causing inappropriate expression orproliferation of cellular genes: for example, genes associated with cellcycle regulation; inflammatory processes; smooth muscle cell (SMC)proliferation, migration and matrix formation (Liu et al., Circulation,79:1374-1387, 1989); certain genetic disorders; and cancers(protooncogenes).

[0076] In one embodiment, translation or RNA processing ofinappropriately expressed cellular genes is blocked. Exemplary potentialtarget sequences are protooncogenes, for example, including but notlimited to the following: c-myc, c-myb, c-fos, c-kit, ras, and BCR/ABL(e.g., Wickstrom, Editor, Prospects for Antisense Nucleic Acid Therapyof Cancer and AIDS, Wiley-Liss, New York, N.Y., 1991; Zalewski et al.,Circulation Res, 88:1190-1195, 1993; Calabretta et al., Seminars inCancer Biol., 3:391-398, 1992; Calabretta et al., Cancer Treatment Rev.19:169-179, 1993), oncogenes/tumor suppresser genes (e.g., p53, Bayeveret al. Antisense Research and Development, 3:383-390, 1993),transcription factors (e.g., NF.kappa.B, Cogswell et al., J. Immunol.,150:2794-2804, 1993) and viral genes (e.g., papillomaviruses, Cowsert etal. Antimicrob. Agents and Chemo., 37:171-177, 1993; herpes simplexvirus, Kulka et al. Antiviral Res., 20:115-130, 1993). To furtherillustrate, two RNA regions of the HIV-1 protein that can be targeted bythe methods of the present invention are the REV-protein responseelement (RRE) and the TAT-protein transactivation response element(TAR). REV activity requires the presence of the REV response element(RRE), located in the HIV envelope gene (Malim et al., Nature,338:254-257, 1989; Malim et al., Cell, 58:205-214, 1989).

[0077] The RRE has been mapped to a 234-nucleotide region thought toform four stem-loop structures and one branched stem-loop structure(Malim et al., Nature, 338:254-257, 1989). Data obtained fromfootprinting studies (Holland et al., J. Virol., 64:5966-5975, 1990;Kjems et al., Proc. Natl. Acad. Sci., 88:683-687, 1991) suggest that REVbinds to six base pairs in one stem structure and to three nucleotidesin an adjacent stem-loop structure of the RRE. A minimum REV bindingregion of about 40 nucleotides in stem-loop II has been identified byCook et al. (Nucleic Acids Research, 19:1577-1583). This binding regioncan be target for generation of RNA/DNA duplexes (e.g., Li et al., J.Virol., 67:6882-6888, 1993) using one or more phosphoramidate2′-arabino-fluorooligonucleotides, according to the methods of thepresent invention.

[0078] The HIV-1 TAT is essential for viral replication and is a potenttransactivator of long terminal repeat (LTR)-directed viral geneexpression (Dayton et al., Cell, 44:941-947, 1986; Fisher et al.,Nature, 320:367-371, 1986). Transactivation induced by TAT proteinrequires the presence of the TAR element (see U.S. Pat. No. 5,837,835)which is located in the untranslated 5′ end of the viral mRNA element.

[0079] The TAR element is capable of forming a stable stem-loopstructure (Muesing et al., Cell, 48:691-701, 1987). The integrity of thestem and a 3 nucleotide (nt) bulge on the stem of TAR has beendemonstrated to be essential for specific and high-affinity binding ofthe TAT protein to the TAR element (Roy et al., Genes Dev., 4:1365-1373,1990; Cordingley et al., Proc. Natl. Acad. Sci., 87:8985-8989, 1990;Dingwall et al., Proc. Natl. Acad. Sci., 86:6925-6929, 1989; Weeks etal., Science, 249:1281-1285, 1990). This region can be targeted foranti-sense therapy following the method of the present invention.

[0080] In addition to targeting the RNA binding sites of the REV, RREand TAT proteins, the RNA coding sequences for the REV and TAT proteinsthemselves can be targeted in order to block expression of the proteins.

[0081] Initial screening of N3′→P5′ phosphoramidate2′-arabino-fluorooligonucleotides, directed to bind potential antisensetarget sites, typically includes testing for the thermal stability ofresultant RNA/DNA duplexes. When a phosphoramidate2′-arabino-fluorooligonucleotide is identified that binds a selected RNAtarget sequence, the oligonucleotide is further tested for inhibition ofRNA function in vitro. Cell culture assays systems are used for such invitro analysis (e.g., herpes simplex virus, Kulka et al., AntiviralRes., 20:115-130, 1993; HIV-1, Li et al., J. Virol., 67:6882-6888, 1993;Vickers et al., Nucleic Acids Research, 19:3359-3368, 1991; coronarysmooth muscle cell proliferation in restenosis, Zalewski et al., NucleicAcids Research, 15:1699-1715, 1987; IL-2R, Grigoriev et al., Proc. Natl.Acad. Sci., 90:3501-3505, 1993; c-myb, Baer et al., Blood, 79:1319-1326,1992; c-fos, Cutry et al., J. Biol. Chem., 264:19700-19705, 1989;BCR/ABL, Szczylik et al., Science, 253:562-565, 1991).

[0082] C. Anti-Gene Applications

[0083] Inhibition of gene expression via triplex formation has beenpreviously demonstrated (Cooney et al., Science, 241:456-459, 1989;Orson et al., Nucleic Acids Research, 19:3435-3441, 1991; Postel et al.,Proc. Natl. Acad. Sci., 88:8227-8231, 1991). The increased stability oftriplex structures formed when employing third strand phosphoramidate2′-arabino-fluorooligonucleotides provides a stronger tool for anti-geneapplications, including veterinary and human therapeutic applications.

[0084] A target region of choice is selected based on known sequencesusing standard rules for triplex formation (Helene and Toulme, Biochem.Biophys. Acta, 1049:99-125, 1990). Typically, the phosphoramidate2′-arabino-fluorooligonucleotide sequence is targeted againstdouble-stranded genetic sequences in which one strand containspredominantly purines and the other strand contains predominantlypyrimidines.

[0085] Phosphoramidate 2′-arabino-fluorooligonucleotides of the presentinvention are tested for triplex formation against a selected duplextarget sequences using band shift assays (see for example, U.S. Pat. No.5,726,297, Example 4). Typically, high percentage polyacrylamide gelsare used for band-shift analysis and the levels of denaturing conditions(Ausubel et al., Current Protocols in Molecular Biology, Hohn Wiley andSons, Inc. Media Pa.; Sauer et al. eds., Methods in EnzymologyProtein/DNA Interactions, Academic Press, 1991; Sambrook et al. inMolecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Vol.2, 1989) are adjusted to reduce any non-specific background binding.

[0086] The duplex target is labeled (for example, using a radioactivenucleotide) and mixed with a third strand oligonucleotide, being testedfor its ability to form triplex structures with the target duplex. Ashift of the mobility of the labeled duplex oligonucleotide indicatesthe ability of the oligonucleotide to form triplex structures.

[0087] Triplex formation is indicated in the band shift assay by adecreased mobility in the gel of the labeled triplex structure relativeto the labeled duplex structure.

[0088] Numerous potential target sites can be evaluated by this methodincluding target sites selected from a full range of DNA sequences thatvary in length as well as complexity. Sequence-specific phosphoramidate2′-arabino-fluorooligonucleotide molecules are potentially powerfultherapeutics for essentially any disease or condition that in some wayinvolves DNA. Exemplary target sequences for such therapeutics include:a) DNA sequences involved in the propagation and/or maintenanceinfectious agents, such as, bacterial, viruses, yeast and other fungi,for example, disrupting the metabolism of an infectious agent; and b)sequences causing inappropriate expression or proliferation of cellulargenes, such as oncogenes, for example, blocking or reducing thetranscription of inappropriately expressed cellular genes (such as genesassociated with certain genetic disorders).

[0089] Gene expression or replication can be blocked by generatingtriplex structures in regions to which required regulatory proteins (ormolecules) are known to bind (for example, HIV transcription associatedfactors like promoter initiation sites and SP1 binding sites, McShan etal., J. Biol. Chem., 267:5712-5721, 1992). Alternatively, specificsequences within protein-coding regions of genes (e.g., oncogenes) canbe targeted as well.

[0090] When a phosphoramidate 2′-arabino-fluorooligonucleotide isidentified that binds a selected duplex target sequence tests, forexample, by the gel band shift mobility assay described above, theanalog is further tested for its ability to form stable triplexstructures in vitro. Cell culture and in vivo assay systems, such asthose described in U.S. Pat. No. 5,631,135 are used.

[0091] Target sites can be chosen in the control region of the genes,e.g., in the transcription initiation site or binding regions ofregulatory proteins (Helene and Toulme, 1990; Birg et al., 1990; Postelet al., 1991; Cooney et al., 1988). Also, target sites can be chosensuch that the target also exists in mRNA sequences (i.e., a transcribedsequence), allowing oligonucleotides directed against the site tofunction as antisense mediators as well (see above).

[0092] Also, phosphoramidate 2′-arabino-fluorooligonucleotide moleculescan be used to generate triplex molecules with a third strand target(i.e., a single-strand nucleic acid). For example, a DNA molecule havingtwo regions capable of forming a triplex structure with a selectedtarget third strand molecule can be synthesized. Typically the tworegions are linked by a flexible region which allows the association ofthe two regions with the third strand to form a triplex.

[0093] Hinge regions can comprise any flexible linkage that keeps thetwo triplex forming regions together and allows them to associate withthe third strand to form the triplex. Third strand targets are selectedto have appropriate purine/pyrimidine content so as to allow formationof triplex molecules.

[0094] The flexible linkage may connect the two triplex forming regions(typically, complementary DNA strands) in any selected orientationdepending on the nature of the base sequence of the target. For example,the two triplex forming regions each have 5′ and 3′ ends, these ends canbe connected by the flexible hinge region in the following orientations:5′ to 3′, 3′ to 5′, 3′ to 3′, and 5′ to 5′.

[0095] Further, duplex DNA molecules containing at least onephosphoramidate 2′-arabino-fluoro nucleotide in each strand can be usedas decoy molecules for transcription factors or DNA binding proteins(e.g., c-myb).

[0096] Single-stranded DNA can also be used as a target nucleic acid foroligonucleotides of the present invention, using, for example,phosphoramidate 2′-arabino-fluorooligonucleotide-containing hairpinstructures. Two phosphoramidate 2′-arabino-fluorooligonucleotides can beselected for single-strand DNA target-directed binding. Binding of thetwo phosphoramidate 2′-arabino-fluorooligonucleotides to thesingle-strand DNA target results in formation of a triplex.

[0097] D. Pharmaceutical Compositions

[0098] The present invention includes pharmaceutical compositions usefulin antisense and antigene therapies. The compositions comprise aneffective amount of N3′→P5′ phosphoramidate2′-arabino-fluorooligonucleotides in combination with a pharmaceuticallyacceptable carrier. One or more N3′→P5′ phosphoramidate2′-arabino-fluorooligonucleotides (having different base sequences) maybe included in any given formulation. In addition, the2′-arabino-fluorooligonucleotides of the present invention may also beused in combination with one or more other oligonucleotides that lack2′-arabino-fluoro analog phosphoramidate nucleosides.

[0099] The N3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotides,when employed in therapeutic applications, can be formulated neat orwith the addition of a pharmaceutical carrier. The pharmaceuticalcarrier may be solid or liquid. The formulation is then administered ina therapeutically effective dose to a subject in need thereof.

[0100] Liquid carriers can be used in the preparation of solutions,emulsions, suspensions and pressurized compositions. The N3′→P5′phosphoramidate 2′-arabino-fluorooligonucleotides are dissolved orsuspended in a pharmaceutically acceptable liquid carrier such as water,an organic solvent, a mixture of both, or pharmaceutically accepted oilsor fats. The liquid carrier can contain other suitable pharmaceuticaladditives including, but not limited to, the following: solubilizers,suspending agents, emulsifiers, buffers, thickening agents, colors,viscosity regulators, preservatives, stabilizers and osmolarityregulators. Suitable examples of liquid carriers for parenteraladministration of N3′→P5′ phosphoramidate2′-arabino-fluorooligonucleotides preparations include water (partiallycontaining additives, e.g., cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g., glycols) and their derivatives,and oils (e.g., fractionated coconut oil and arachis oil).

[0101] For parenteral administration of N3′→P5′ phosphoramidate2′-arabino-fluorooligonucleotides the carrier can also be an oily estersuch as ethyl oleate and isopropyl myristate. Sterile carriers areuseful in sterile liquid form compositions for parenteraladministration.

[0102] Sterile liquid pharmaceutical compositions, solutions orsuspensions can be utilized by, for example, intraperitoneal injection,subcutaneous injection, intravenously, or topically. For example,antisense oligonucleotides directed against retinal cytomegalovirusinfection may be administered topically by eyedrops. N3′→P5′phosphoramidate 2′-arabino-fluorooligonucleotides can be also beadministered intravascularly or via a vascular stent impregnated withmycophenolic acid, for example, during balloon catheterization toprovide localized anti-restenosis effects immediately following injury.

[0103] The liquid carrier for pressurized compositions can behalogenated hydrocarbon or other pharmaceutically acceptable propellant.Such pressurized compositions may also be lipid encapsulated fordelivery via inhalation. For administration by intranasal orintrabronchial inhalation or insufflation, N3′ P5′ phosphoramidate2′-arabino-fluorooligonucleotides may be formulated into an aqueous orpartially aqueous solution, which can then be utilized in the form of anaerosol, for example, for treatment of infections of the lungs likePneumocystis carnil.

[0104] N3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotides may beadministered topically as a solution, cream, or lotion, by formulationwith pharmaceutically acceptable vehicles containing the activecompound. For example, for the treatment of genital warts.

[0105] The N3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotides maybe administered in liposome carriers. The use of liposomes to facilitatecellular uptake is described, for example, in U.S. Pat. Nos. 4,897,355(D. Eppstein et al., issued 30 Jan. 1990) and U.S. Pat. No. 4,394,448(F. Szoka et al., issued 19 Jul. 1983). Numerous publications describethe formulation and preparation of liposomes.

[0106] The dosage requirements for treatment with N3′→P5′phosphoramidate 2′-arabino-fluorooligonucleotides vary with theparticular compositions employed, the route of administration, theseverity of the symptoms presented, the form of N3′→P5′ phosphoramidate2′-arabino-fluorooligonucleotides and the particular subject beingtreated.

[0107] In general, N3′ P5′ phosphoramidate2′-arabino-fluorooligonucleotides are administered at a concentrationthat affords effective results without causing any harmful ordeleterious side effects (e.g., an effective amount). Such aconcentration can be achieved by administration of either a single unitdose, or by the administration of the dose divided into convenientsubunits at suitable intervals throughout the day.

[0108] E. Diagnostic Applications

[0109] The phosphoramidate 2′-arabino-fluorooligonucleotides of thepresent invention are also useful in diagnostic assays for detection ofRNA or DNA having a given target sequence. In one general application,the phosphoramidate 2′-arabino-fluorooligonucleotides are labeled (e.g.,isotopically or other detectable reporter group) and used as probes fornucleic acid samples that bound to a solid support (e.g., nylonmembranes).

[0110] Alternatively, the phosphoramidate2′-arabino-fluorooligonucleotides may be bound to a solid support (forexample, magnetic beads) and homologous RNA or DNA molecules in a sampleseparated from other components of the sample based on theirhybridization to the immobilized phosphoramidate analogs. Binding ofphosphoramidate 2′-arabino-fluorooligonucleotides to a solid support canbe carried out by conventional methods. Presence of the bound RNA or DNAcan be detected by standard methods, for example, using a second labeledreporter or polymerase chain reaction (see U.S. Pat. Nos. 4,683,195 and4,683,202).

[0111] Diagnostic assays can be carried out according to standardprocedures, with suitable adjustment of the hybridization conditions toallow phosphoramidate 2′-arabino-fluorooligonucleotide hybridization tothe target region. The ability of phosphoramidate2′-arabino-fluorooligonucleotides to bind at elevated temperature canalso help minimizes competition for binding to a target sequence betweenthe phosphoramidate 2′-arabino-fluorooligonucleotides probe and anycorresponding single-strand phosphodiester oligonucleotide that ispresent in the diagnostic sample.

[0112] F. Other Applications

[0113] In one aspect, the phosphoramidate2′-arabino-fluorooligonucleotides can be used in methods to enhanceisolation of RNA or DNA from samples. For example, as discussed above,phosphoramidate 2′-arabino-fluorooligonucleotides can be fixed to asolid support and used to isolate complementary nucleic acid sequences,for example, purification of a specific mRNA from a polyA fraction(Goldberg et al., Methods in Enzymology, 68:206, 1979). Thephosphoramidate 2′-arabino-fluorooligonucleotides are advantageous forsuch applications since they can form more stable interactions with RNAand duplex DNA than standard phosphodiester oligonucleotides.

[0114] A large number of applications in molecular biology can be foundfor reporter labeled phosphoramidate 2′-arabino-fluorooligonucleotides,particularly for the detection of RNA in samples. Phosphoramidate2′-arabino-fluorooligonucleotides can be labeled with radioactivereporters (3H, ¹⁴C, ³²P, or ³⁵S nucleosides), biotin or fluorescentlabels (Gryaznov et al., Nucleic Acids Research, 20:3403-3409, 1992).Labeled phosphoramidate 2′-arabino-fluorooligonucleotides can be used asefficient probes in, for example, RNA hybridization reactions (Ausubelet al., Current Protocols in Molecular Biology, Hohn Wiley and Sons,Inc., Media, Pa.; Sambrook et al., in Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Vol. 2, 1989).

[0115] Also, double-stranded DNA molecules where each strand contains atleast one phosphoramidate 2′-arabino-fluoronucleotide can be used forthe isolation of DNA-duplex binding proteins. In this embodiment aduplex containing phosphoramidate 2′-arabino-fluorooligonucleotide istypically affixed to a solid support and sample containing a suspectedbinding protein is then passed over the support under buffer conditionsthat facilitate the binding of the protein to its DNA target. Theprotein is typically eluted from the column by changing bufferconditions.

[0116] The triplex forming DNA molecules described above, containingphosphoramidate 2′-arabino-fluorooligonucleotides, can be used asdiagnostic reagents as well, to, for example, detect the presence of anRNA molecule in a sample.

[0117] Further, complexes containing oligonucleotides having N3′→P5′phosphoramidate 2′-arabino-fluoro nucleotides can be used to screen foruseful small molecules or binding proteins: for example, N3′→P5′phosphoramidate 2′-arabino-fluorooligonucleotide complexes with duplexDNA can be used to screen for small molecules capable of furtherstabilizing the triplex structure. Similar screens are useful withN3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotide complexesformed with single strand DNA and RNA molecules.

[0118] G. Variations

[0119] Variations on the phosphoramidate2′-arabino-fluorooligonucleotides used in the methods of the presentinvention include modifications to facilitate uptake of theoligonucleotide by the cell (e.g., the addition of a cholesterol moiety(Letsinger, U.S. Pat. No. 4,958,013); production of chimericoligonucleotides using other intersubunit linkages (Goodchild,Bioconjugate Chem., 1:165-187, 1990); modification with intercalatingagents (for example, triplex stabilizing intercalating agents, Wilson etal., Biochemistry, 32:10614-10621, 1993); and the use of ribose insteadof deoxyribose subunits. Further modifications include, 5′ and 3′terminal modifications to the oligonucleotides (e.g., —OH, —OR, —NHR,NH₂ and cholesterol).

[0120] N3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotides mayalso be modified by conjugation to a polypeptide that is taken up byspecific cells. Such useful polypeptides include peptide hormones,antigens and antibodies. For example, a polypeptide can be selected thatis specifically taken up by a neoplastic cell, resulting in specificdelivery of N3′→P5′ phosphoramidate 2′-arabino-fluorooligonucleotides tothat cell type. The polypeptide and oligonucleotide can be coupled bymeans known in the art (see, for example, PCT International ApplicationPublication No. PCT/US89/02363, WO8912110, published Dec. 14, 1989,Ramachandr, K. et al.).

[0121] The properties of such modified phosphoramidate2′-arabino-fluorooligonucleotides, when applied to the methods of thepresent invention, can be determined by the methods described herein.

EXAMPLE 1 General Methods

[0122]³¹p NMR spectra were obtained on a Varian 400 MHz spectrometer.³¹p NMR spectra were referenced against 85% aqueous phosphoric acid.Anion exchange HPLC was performed using a Dionex DX 500 ChromatographySystem, with a Pharmacia Biotech Mono Q HR 5/5 or 10/16 ion exchangecolumns. Ion exchange HPLC was performed on the Dionex DX 500 system,using a Pharmacia Mono Q HR 5/5 column. Buffer A and B were: 10 mM NaOH,pH 12 and 10 mM NaOH, 1.5 M NaCl, pH 12, respectively. Oligonucleotideswere eluted using a 1.5%/min. linear gradient of buffer B in A; flow 0.5mL/min. Mass spectral analysis was performed by Mass Consortium, SanDiego, Calif. MALDI-TOF analysis of oligonucleotides was obtained usinga PerSpective Biosystems Voyager Elite mass spectrometer with delayedextraction. Thermal dissociation experiments were conducted on a CaryBio 100 UV-Vis spectrometer.

[0123] All reactions were carried out in oven dried glassware under anitrogen atmosphere unless otherwise stated. Commercially available DNAsynthesis reagents were purchased from Glen Research (Sterling, Va.).Anhydrous pyridine, toluene, dichloromethane, diisopropylethyl amine,triethylamine, acetic anhydride, 1,2-dichloroethane, and dioxane werepurchased from Aldrich (Milwaukee, Wis.).

[0124] All non-2′-arabino-fluorooligonucleotides were synthesized on anABI 392 or 394 DNA synthesizer using standard protocols for thephosphoramidite based coupling approach (Caruthers, Acc. Chem. Res.,24:278-284, 1991). The chain assembly cycle for the synthesis ofoligonucleotide phosphoramidates was the following: (i) detritylation,3% trichloroacetic acid in dichloromethane, 1 min.; (ii) coupling, 0.1 Mphosphoramidite and 0.45 M tetrazole in acetonitrile, 1-3 min.; (iii)capping, 0.5 M isobutyic anhydride in THF/lutidine, 1/1, v/v, 15 sec;and (iv) oxidation, 0.1 M iodine in THF/pyridine/water, 10/10/1, v/v/v,30 sec.

[0125] Chemical steps within the cycle were followed by acetonitrilewashing and flushing with dry argon for 0.2-0.4 min. Cleavage from thesupport and removal of base and phosphoramidate protecting groups wasachieved by treatment with ammonia/EtOH, 3/1, v/v, for 6 h at 55° C. Theoligonucleotides were concentrated to dryness in vacuo after which the2′-t-butyldimethylsilyl groups were removed by treatment with 1M TBAF inTHF for 4-16 h at 25° C. The reaction mixtures were diluted with waterand filtered through a 0.45 nylon acrodisc (from Gelman Sciences, AnnArbor, Mich.). Oligonucleotides were then analyzed and purified by IonExchange HPLC and finally desalted using gel filtration on a PharmaciaNAP-5 or NAP-25 column.

EXAMPLE 2 Synthesis of 2′-Arabino-Fluorornucleoside PhosphoramiditeMonomers

[0126] Preparation of the inventive 2′-arabino-fluorornucleosidemonomers is described in FIG. 1. First, a1-α-O-Benzoyl-3,5-O-benzoyl-2-arabinofluoro-2-deoxyfuranose sugarprecursor 1 (FIG. 1) (from Pfanstiehl Laboratories, Inc., Waukegan,Ill.) was converted into a1-α-Bromo-3,5-O-benzoyl-2-arabinofluoro-2-deoxyfuranose intermediate 2with retention of sugar C-1 configuration (Berger et al., Nucl. AcidsRes., 26:2473-2480, 1998). Compound 2 was used, without isolation, in aS_(N)2-type glycosylation reaction with silylated purine and pyrimidinebases according to the literature procedure (Berger et al., Nucl. AcidsRes., 26:2473-2480, 1998), which resulted in formation of2′-arabinofluoro-3′,5′-O-benzoyl nucleosides 3 (FIG. 1). Stereoselectivity of this glycosylation reaction was quite high-more than 90%of the formed nucleoside 3 had the desired β-anomeric configuration, aswas judged by ¹H NMR analysis of crystallized products. Then, 5′- and3′-O-benzoyl protecting groups of nucleoside 3 were removed, almostquantitatively, by methanolic ammonia, and the resultant 5′-,3′-hydroxyl groups containing nucleoside product without additionalpurification was converted into2,3′-anhydro-2′-arabinofluoro-5′-O-benzoyl nucleosides 4 under Mitsunobureaction conditions (Czernecki et al., Synthesis, 239-240, 1991). Thefollowing treatment of the -2,3′-anhydronucleosides with lithium azideresulted in key 2′-arabinofluoro-3′-azido-5′-O-benzoyl precursor 5(Glinski, et al., J. Chem. Soc. Chem. Comm., 915-916, 1970). Thesecompounds were converted into thymidine and uracil2′-arabinofluoro-3′-NH-MMT-5′-O-(cyanoethyl-N,N′-diisopropylamino)-phosphoramidites7 as described in the literature sequence of chemical transformations:catalytic reduction of 3′-azido to 3′-amino group by hydrogen overpalladium, followed by 3′-tritylation, 5′-O-deprotection and5′-O-phosphitylation (Schultz et al., Nucl. Acids Res., 24:2966-2973,1996). Cytosine phosphoramidite 7 c was obtained from the 3′-azidoprecursor uracil-to-cytosine conversion process analogous to theliterature procedure for 3′-azido-2′-ribo-fluoronucleosides (Schultz etal., Nucl. Acids Res., 24:2966-2973, 1996) (FIG. 1). Total yields of thecytosine, thymidine and uracil phosphoramidites 7 c,u,t were in therange of 15-20% as calculated based on the starting sugar precursor 1.Structure of the monomers was confirmed by ¹H, ³¹P, ¹⁹F NMR and by massspectrometric analysis. For example, for thymidine 7, Rp-, Sp-isomers,³¹P NMR, L, PPM (CDCl₃) 149.4; 149.8 ¹⁹F NMR L, ppm (CDCl₃) two hextetsat −190.26 and −190.88). The data for uracil 7 and cystosine 7 is verysimilar: ³¹P NMR, L, PPM (CDCl₃) in range of 148-149; ¹⁹F NMR L, ppm(CDCl₃) two hextets at about −188 and −191.

[0127] 2′-Arabino-fluoro-amino purine monomers can also be preparedstarting with purine-3′-azido-2′-hydroxylprecursor, or purine3′-NH-trityl-2′-hydroxyl precursors that are prepared using thefollowing synthesis methods. The first step of the synthesis involvedtin(IV) chloride or trimethylsilyl triflate mediated glycosylation oftrimethylsilylated nucleobases (Azhayev, et al. (1979) Nucleic AcidsRes., 2:2625-2643; Vorbruggen, et al. (1981) Chem. Ber., 114:1234-1255)to a commonly employed sugar precursor3-azido-1,2-di-O-acetyl-5-O-toluoyl-3-deoxy-D-ribofuranose 10, which wasprepared according to literature procedure (Ozols, et al. Synthesis,557-558). Adenine was protected at N⁶ with a benzoyl group, whileguanine was blocked at N² with an isobutyl group and at O⁶ withdiphenylcarbamate (Zou, et al. (1987) Can. J. Chem., 65:1436-1437). Theprotection of O⁶ with this bulky group allows for selectiveglycosylation to occur at N⁹ with very little (<10%) formation of theundesired N⁷ regioisomer as judged by TLC analysis. 2,6-Diaminopurinewas protected at each exocylic amine with a phenoxyacetyl group for allglycosylation reactions with this highly polar purine base analogue(Schulhof, et al. (1987) Tetrahedron Lett., 28:51-54).

[0128] Two key synthetic improvements were made to the preparation ofthe monomers, which allowed for rapid access to the final products withimproved overall yields. First, experimental conditions were found whichenabled selective removal of the 2′-O-acetyl protecting group in thepresence of the 5′-O-toluoyl counterpart (Neilson, et al. (1971) Can. J.Chem., 49:493-498) (FIG. 2). This allowed for the omission of a5′-hydroxyl reprotection step from the synthetic protocol. Also, a lowyielding series of steps late in the monomer synthesis, used in theliterature procedure (Gryaznov, et al. (1998) Nucleic Acids Res.,26:4160-4167) to convert a 5′-O-trityl-nucleoside precursor to the3′-N-trityl-protected amino intermediate, was also averted. Secondly,following the glycosylation reaction, the next five chemicaltransformations were conducted with very high yields. This eliminatedthe need for intermediate purification after steps iv.-viii. (FIG. 2),thus providing a rapid and convenient access to compounds 17 a-dHowever, it should be noted that for the guanosine and 2,6-diaminopurineanalogues, selective removal of the 2′-O-acetyl protecting group wasunsuccessful. Thus, both 2′-O- and 5′-O-protecting groups were removed,after which the 5′-hydroxyl group was selectively reprotected (FIG. 2).

[0129] For compound 11 a the 2′-O-acetyl group was selectively removedusing 50% (v/v) aqueous ammonia in methanol followed by the 3′-azidogroup reduction with hydrogen over palladium on carbon. Each of thesereactions proceeded with very high, near quantitative, yields as judgedby TLC and ¹H NMR analysis of the products. The 3′-amino group is thenprotected by treatment with 4-monomethoxytrityl chloride to givecompound 15 a. The obtained protected nucleoside precursor was thentreated with diethylamino sulfur trifluoride (DAST) to make anarabino-fluoro adenine nucleoside 16 a. The protected arabino-fluoroproduct is concentrated in vacuo to which is added 1.0 M NaOH in 65/30/5pyridine/MeOH/H₂O (70 mL) at 0° C. to remove the 5′-O-toluoyl group. Themixture is stirred for 8 min and quenched by addition of saturatedNH₄Cl. This solution is extracted with ethyl acetate (2×75 mL) and thecombined organic layers are dried over Na₂SO₄, filtered, andconcentrated in vacuo. The residue is purified by silica gelchromatography eluting with EtOAc:hexanes (50:50, v/v) to yield2′-arabino-fluorornucleoside 107 a. In an alternative method, the fluoroaddition is performed prior to reduction of the 2′ azido group to anamino group (e.g. prior to step v. in FIG. 2).

[0130] The inability to selectively remove the 2′-O-acetyl group fromintermediates 11 g and 11 d, necessitated the following syntheticprotocol. Both 2′-O- and 5′-O protecting groups were removed with 1 Msodium hydroxide, after which a 5′-O-anisoyl group was selectivelyreintroduced under Mitsunobu conditions to give 13 g and 13 d. Compounds11 g and 11 d (about 2.8 g, 3.81 mmol) are dissolved in a 1.0 M NaOHsolution (65/30/5 pyridine/MeOH/H₂O, v/v/v, (40 mL)) at 0° C. Themixtures are stirred for 10 min, and then quenched by addition ofsaturated NH₄Cl (400 mL). The solutions are extracted with CH₂Cl₂ (5×100mL) and the combined organic layers are dried over Na₂SO₄, filtered, andconcentrated in vacuo. For guanine nucleosides the residues istriturated well with Et₂OH (50 mL) to remove diphenylamine and theresulting material dissolved in acetonitrile (25 mL) andtriphenylphosphine (1.2 g, 4.65 mmol). For diaminopurine nucleosides theNa₂SO₄ dried, filtered and concentrated material is dissolvedindimethylformamide (50 ml) and triphenylphosphine (1.5 g, 5.7 mmol) areadded. For the remainder of the steps leading to 13 g,d the steps arethe same. p-Anisic acid (0.71 g, 4.65 mmol) and diisopropylazodicarboxylate (0.92 mL, 4.65 mmol) are dissolved in acetonitrile (5mL) and added dropwise to the reaction mixtures. The solutions arestirred at room temperature for 1 h and was then quenched by pouringthem into saturated NaHCO₃ (200 mL). The mixtures are extracted withethyl acetate (200 mL) and after separation the organic phases arewashed with saturated NaCl (150 mL). The organic phases are then driedover Na₂SO₄ and concentrated in vacuo. The residues are purified bysilica gel chromatography eluting with a gradient of EtOAc:Hexanes:MeOH(49:49:2, v/v/v to 47.5:47.5:5, v/v/v) to afford 13 g,d.

[0131] It should be noted that the high reactivity of the 2′-hydroxylgroup of the 3′-azido-2′-hydroxyl guanosine intermediate preventedselective reprotection of the 5′-hydroxyl group by either benzoylchloride or benzoyl anhydride. The same series of steps described abovefor adinine are used to convert 13 g and 13 d into 17 g and 17 drespectively (FIG. 2). The MMT protecting group in 17 a-d can be removedto give the unprotected amino sugar moiety. The 5′-OH group can then beselecteively protected with an alkyl group or converted to a mono, di,or triphosphate group. The final step for monomer preparation involvesphosphitylation of 17 a-d to give the5′-(2-cyanoethyl-N,N′-diisopropylamino)nucleoside phosphoramiditebuilding blocks 18 a-d (FIG. 2).

[0132] In order to gain information on the2′-arabino-fluoro-3′-aminonucleosides sugar puckering, a model N3′→P5′phosphoramidate tri-nucleotide dTn-a(U^(f)nU^(f)n) [SEQ ID NO:1],containing internucleoside and terminal2′-arabino-fluoro-3′-aminonucleosides (³¹P NMR,L,ppm in D₂O 7.8 and 6.7)was synthesized, and the compound was analyzed by high resolution ¹H and³¹P NMR spectroscopy. The analysis revealed vicinal proton couplingconstants J³H1′-H2″ of 4.68 Hz for both 2′-arabino-fluoro-uridinenucleosides, and J³H2″-H3′ 3.81 Hz and 4.46 Hz for internucleoside andfor 3′-terminal 2′-arabino-fluoro-uridines, respectively. Thecorresponding coupling constants for the prepared model di-nucleosideau^(f)pdT [SEQ ID NO:2], containing 2′-arabino-fluoro-3′-hydroxy uraciland the phosphodiester internucleoside bond, were 3.67 Hz and 1.83 Hzrespectively, which indicates a significant difference in 3′-NH— and3′-O— 2′-ara-fluoronucleosides sugar puckering. The observed couplingconstants for the tri-nucleotide alone do not define the2′-arabino-fluoro-3′-amino sugars conformation. However, the dataobtained from 2D COSY spectra of the tri-nucleotide large intensity ofH3′-H4′ cross peaks, as well as the data from 1H-³¹P 2D heteronuclearspectra (strong H3′-(i−1)P and weak H4′-P cross peaks) suggestprevalence of N-type conformation for the2′-arabino-fluoro-3′-aminonucleosides. The substitution of 3′-oxygen by3′-amino group apparently shifts the 2′-arabino-fluoro furanoseconformational equilibrium towards N-type, or C3′-endo, for2′-arabino-fluoronucleosides, which is similar to that for the2′-deoxy-3′-aminonucleosides.

EXAMPLE 3 Synthesis of N3′→P5′ Phosphoramidite2′-Arabino-Fluorooligonucleotides

[0133] The oligo-2′-arabino-fluoro nucleotide N3′→P5′ phosphoramidateswere assembled similarly to the oligo-2′-ribo-fluoro N3→′P5′phosphoramidate compounds. Solid phase synthesis was based on thephosphoramidite transfer reaction using monomer building blocks composedof 5′-(O-cyanoethyl-N,N′-diisopropylamino)-phosphoramidites of3′-MMTr-protected-3′-amino-2′-arabino-fluoronucleosides (Schultz et al.,Nucl. Acids Res., 24:2966-2973, 1996).

[0134] The oligonucleotide synthesis was conducted on an automatedDNA/RNA ABI 394 synthesizer using the synthetic cycle described beforefor the 2′-ribo-fluoro phosphoramidites with step-wise coupling yieldsof about 95-97%. Each of the prepared 2′-arabino-fluorooligonucleotideswere synthesized, starting from the 5′-end using a support-bound2′-deoxy-3′-aminonucleoside as the 5′-terminal residue. Coiling stepsinvolved exchange of the diisopropylamino group of the approaching5′-O-phosphoramidite monomer for the 3′-amino group of the support boundnucleoside. Standard RNA synthesis coupling times (10 min) and activator(1H-tetrazole) were used for each synthetic cycle. Unreacted 3′-aminogroups were then capped with isobutyric anhydride, after which oxidationof the internucleotide phosphoramidite diester linkage into thephosphoramidate group was carried out with aqueous iodine. Subsequentdetritylation of the 3′-amino group of the added residue enabledadditional chain elongation steps to be repeated for the construction ofthe desired 2′-arabino-fluororooligonucleotide phosphoramidates.

[0135] Compounds were cleaved from a solid phase support and all theprotective groups were removed with concentrated aqueous ammonia, about0.5 to 4.5 hours at 55° C. The resulting2′-arabino-fluorooligonucleotide N3′→P5′ phosphoramidates have thestructure shown in FIG. 3. The inventive oligonucleotide structure shownin FIG. 3 was determined by first purifying and then analyzing thecleaved and deprotected reaction mixtures. The synthesized oligos werepurified by ion exchange (IE) HPLC. The structure of the IE HPLCisolated oligo-2′-arabino-fluoronucleotide N3′→P5′ phosphoramidates wasconfirmed by ³¹P and ¹⁹F NMR analysis.

[0136] The sequences of representative inventive oligonucleotides thathave been prepared are summarized in Table 1. TABLE 1 OLIGONUCLEOTIDESAND MELTING TEMPERATURE (Tm) VALUES OF THEIR COMPLEXES T_(m), ° C.^(b)T_(m), ° C.^(b) Expt. 5′-Oligonucleotide-3′^(a) RNA DNA dsDNA^(e) 1d(UpUpUpUpUpUpUpUpUpT) 17.9;^(c) 20.3^(d) 16.7;^(c) 24.6^(d) n.o;^(c)n.o^(d) [SEQ ID NO:3] 2r(Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)T) 32.9; 36.6n.o; 18.0 n.o; n.o [SEQ ID NO:4] 3a(Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)T) 27.5; 28.617.0; 25.0 <14; 20.9 [SEQ ID NO:5] 4 d(UnUnUnUnUnUnUnUnUnT) 38.1; 47.218.5; 38.2 <15; n.d [SEQ ID NO:6] 5dT-r(Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)) 55.5; 61.937.4; 56.3 42.2; 54.1 [SEQ ID NO:7] 6dT-a(Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)Up^(f)) 40.0; 40.025.7; 37.0 22.0; 31.2 [SEQ ID NO:8] 7 d(CnUnCnUnCnUnGnCnCn) 66.7 n.d —[SEQ ID NO:9] 8 a(Cn^(f)Un^(f)Cn^(f)Un^(f)Cn^(f)Un^(f)GnCn^(f)Cn^(f))66.8 n.d — [SEQ ID NO: 10]

[0137] First, the compounds were characterized by ion exchange (IE) HPLCanalysis. Oligo-2′-arabino-fluoronucleotide N3′→P5′ phosphoramidate [SEQID NO:6], Table 1, has significantly longer, by about 8 minutes,retention time on IE HPLC column at pH 12, than that for theisosequential 2′-ribo-fluoro phosphoramidate counterpart oligonucleotide[SEQ ID NO:7], Table 1. At the same time these oligonucleotidespractically co-elute from the same column at pH 7 buffer conditions. IEHPLC was carried out on Dionex DX 500 system, using Pharmacia MonoQ 5/5column; Buffer A: 10 mM NaOH, pH 12; Buffer B: 10 mM NaOH, 1.5 M NaCl,pH 12; 1.5%/min. linear gradient of buffer B in A; flow 0.5 mL/min.Retention time (Rt), for co-injected oligonucleotides SEQ ID NOs:7, 8, 4and 5, Table 1, was 33.6, 41.5, 42.5 and 46.5 minutes, respectively. AtpH 7.2 (10 mM Na-phosphate buffer) retention times for oligonucleotidesSEQ ID NOs:7 and 8 were 23.7 and 23.8 minutes, respectively. Thedifferent degrees of uracil base ionization under the alkalineconditions, as well as ionization of internucleoside 3′-NHP(O)0-5′groups, which are influenced by the spatial orientation of the2′-fluorine likely determine the differences in the oligomerschromatographic behavior, Probably, the 2′-fluorine atom increasesacidity of uracil bases and in trans-oriented internucleosidephosphoramidate groups better in arabino—than in ribo-configuration.Additionally, retention times (at pH 12) for the 2′-ribo-fluoro and2′-arabino-fluoro phosphodiester decanucleotides SEQ ID NOs:4 and 5,Table 1, were noticeably longer, than for their phosphoramidate cognateoligonucleotides SEQ ID NOs:7 and 8, Table 1. This difference likelyreflects a higher lypophilicity of 2′-fluoro phosphodiesteroligonucleotides. Also, similarly to the phosphoramidates,oligo-2′-arabino-fluoronucleotide was retained longer than the2′-ribo-fluoro isomer on IE HPLC column, indicating its highernet-negative charge in alkaline conditions.

EXAMPLE 4 Stability and Duplex Formation Properties of N3′→P5′Phosphoramidate 2′-Arabino-Fluorooligonucleotides

[0138] It was reported that the 2′-ribo-fluoropyrimidine containingoligonucleotides with N3′→P5′ phosphoramidate or with phosphodiesterinternucleoside linkages are somewhat labile during treatment withaqueous ammonia at 55° C. (Schultz et al., Nucl. Acids Res.,24:2966-2973, 1996; Krug et al., Nucleosides Nucleotides, 8:1473-1483,1989). Under these basic conditions pyrimidine-02 mediated eliminationof the 2′-fluorine atom takes place, resulting in formation of2,3′-O-anhydronucleosides and multiple products of their consequentreactions with aqueous ammonia, including 2′-arabino-hydroxylnucleosides (Krug et al., Nucleosides Nucleotides, 8:1473-1483, 1989).In contrast, the prepared 2′-arabino-fluoro phosphoramidateoligonucleotides were stable under these conditions. The2′-arabino-fluoro phosphoramidate decanucletide SEQ ID NO:8, Table 1,was practically intact after exposure to concentrated aqueous ammoniafor 24 hours, 55° C., whereas isomeric 2′-ribo-fluoro counterpart SEQ IDNO:7, Table 1, was converted into a very complex mixture of productsunder these conditions, as was judged by IE HPLC analysis. Moreover, thehalf-life of 2′-arabino-fluoro phosphoramidate SEQ ID NO:8 in acidicmedia, pH 3, was about 2 and 9 times longer (about 280 minutes), thanthat for the isosequential 2′-ribo-fluoro phosphoramidateoligonucleotide SEQ ID NO:7 (about 135 minutes) and for 2′-deoxyphosphoramidate oligonucleotide SEQ ID NO:6 (about 30 minutes),respectively (see Schultz, et al. 1996 for acid stability method).

[0139] The ability of N3′→P5′ phosphoramidate2′-arabino-fluorooligonucleotides to form complexes with complementaryDNA and RNA strands was examined and compared with relatedoligonucleotide analogues using thermal denaturation experiments. Theresults of the study are summarized in Table 1. Substitution of 2′-deoxynucleosides by their 2′-ribo-fluoro cognates in phosphodiesteroligonucleotide, compounds SEQ ID NOs:3 and 4, resulted in significantstabilization of duplexes with RNA—T_(m) of about 15.0-16.3° C., butdestabilization of complexes with DNA—T_(m) of about −6.7-16° C.(compare experiments 1 and 2, Table 1). 2′-arabino-fluoro and 2′-deoxyphosphodiester oligonucleotides SEQ ID NOs:5 and 10 formed duplexes ofsimilar stability with DNA, but, with RNA the T_(m) of the2′-arabino-fluorooligonucleotide complex was about 8.3-9.6° C. higherthan the 2′-deoxy phosphodiester oligonucleotide (compare experiments 1and 3, Table 1). Substitution of 2′-deoxy-3′-aminonucleosides by2′-arabino-fluoro-3′-amino counterparts stabilized duplexes, by about2-7° C., with DNA and RNA in a low ionic strength buffer. In presence ofan additional 10 mM magnesium chloride a 2′-deoxy phosphoramidate duplexwith RNA is more stable, by about 7° C., than the duplex formed by a2′-arabino-fluorooligonucleotide counterpart (compare experiments 4 and6, Table 1). Interestingly, increasing the buffer ionic strengthunexpectedly had no effect on T_(m) value of oligonucleotide SEQ ID NO:6duplex with RNA. Also, 2′-arabino-fluoro phosphoramidate SEQ ID NO:6duplexes were more stable than those formed by a 2′-arabino-fluorophosphodiester oligonucleotide SEQ ID NO:5, −T_(m) 8.7-12.5° C. (compareexperiments 3 and 6, Table 1). The most stable complexes with both DNAand RNA strands, −T_(m) of about 14-21° C., were formed by2′-ribo-fluoro phosphoramidate oligonucleotide SEQ ID NO:7 (compareexperiments 5, 6 and 7, Table 1). Duplexes formed by the mixed base2′-arabino-fluoro and 2′-deoxy phosphoramidate oligonucleotides SEQ IDNOs:9 and 10 have a similar thermal stability, which likely reflectssimilarity of the nucleosides sugar puckering, as indicated by the NMRanalysis (compare experiments 7 and 8, Table 1). Moreover,2′-arabino-fluoro phosphoramidate oligonucleotide SEQ ID NO:8 formed amore stable triplex with dsDNA than did a 2′-deoxy phosphoramidateoligonucleotide SEQ ID NO:6, or phosphodiester oligonucleotides SEQ IDNOs:3-5, but not a 2′-ribo-fluoro counterpart oligonucleotide SEQ IDNO:7 (Table 1). These data demonstrate the synergistic duplex andtriplex stabilizing effects of 2′-arabino-fluoro and 3′-aminomodifications.

EXAMPLE 5 Preparation of Affinity Purified Extract Having TelomeraseActivity

[0140] Extracts used for screening telomerase inhibitors were routinelyprepared from 293 cells over-expressing the protein catalytic subunit oftelomerase (hTERT). These cells were found to have 2-5 fold moretelomerase activity than parental 293 cells. 200 ml of packed cells(harvested from about 100 liters of culture) were resuspended in anequal volume of hypotonic buffer (10 mM Hepes pH 7.9, 1 mM MgCl₂, 1 mMDTT, 20 mM KCl, 1 mM PMSF) and lysed using a dounce homogenizer. Theglycerol concentration was adjusted to 10% and NaCl was slowly added togive a final concentration of 0.3 M. The lysed cells were stirred for 30min. and then pelleted at 100,000×g for 1 hr. Solid ammonium sulfate wasadded to the S100 supernatant to reach 42% saturation. The material wascentrifuged; the pellet was resuspended in one fifth of the originalvolume and dialyzed against Buffer ‘A’ containing 50 mM NaCl. Afterdialysis the extract was centrifuged for 30 min. at 25,000×g. Prior toaffinity chromatography, Triton X-100 (0.5%), KCl (0.3 M) and tRNA (50μg/ml) were added. Affinity oligo (5′ biotinTEG-biotinTEG-biotinTEG-GTAGAC CTG TTA CCA guu agg guu ag 3′ [SEQ ID NO:11]; lower case represents2′ O-methyl ribonucleotides and upper case represents deoxynucleotides)was added to the extract (1 nmol per 10 ml of extract). After anincubation of 10 min. at 30° C., Neutravidin beads (Pierce; 250 μl of a50% suspension) were added and the mixture was rotated overnight at 4°C. The beads were pelleted and washed three times with Buffer ‘B’containing 0.3 M KCl, twice with Buffer ‘B’ containing 0.6 M KCl, andtwice more with Buffer B containing 0.3 M KCl. Telomerase was eluted inBuffer ‘B’ containing 0.3 M KCl, 0.15% Triton X-100 and a 2.5 molarexcess of displacement oligo (5′-CTA ACC CTA ACT GGT AAC AGG TCT AC-3′[SEQ ID NO:12] at 0.5 ml per 125 μl of packed Neutravidin beads) for 30min. at room temperature. A second elution was performed and pooled withthe first. Purified extracts typically had specific activities of 10fmol nucleotides incorporated/min./μl extract, or 200nucleotides/min./mg total protein. Buffer ‘A’ Buffer ‘B’ 20 mM Hepes pH7.9 20 mM Hepes pH 7.9  1 mM MgCl2  1 mM EDTA  1 mM DTT  1 mM DTT  1 mMEGTA  10% glycerol 10% glycerol 0.5% Triton X-100

EXAMPLE 6 Telomerase Inhibition by Oligonucleotide N3′→P5′2′-Arabino-Fluoro Phosphoramidates

[0141] Three separate 100 μl telomerase assays are set up with thefollowing buffer solutions: 50 mM Tris acetate, pH 8.2, 1 mM DTT, 1 mMEGTA, 1 mM MgCl₂, 100 mM K acetate, 500 μM dATP, 500 μM TTP, 10 μM³²P-dGTP (25 Ci/mmol), and 100 nM d(TTAGGG)₃ [SEQ ID NO: 13]. To theindividual reactions 2.5, 5 or 10 μl of affinity-purified telomerase(see Example 5) is added and the reactions are incubated at 37 C. At 45and 90 minutes, 40 μl aliquots are removed from each reaction and addedto 160 μl of Stop Buffer (100 mM NaCl, 10 mM Na pyrophosphate, 0.2% SDS,2 mM EDTA, 100 μg/ml tRNA). 10 μl trichloroacetic acid (TCA) (100%) isadded and the sample is incubated on ice for 30 minutes. The sample ispelleted in a microcentrifuge (12000×g force) for 15 minutes. The pelletis washed with 1 ml 95% ethanol and pelleted again in themicrocentrifuge (12000×g force) for 5 minutes. The pellet is resuspendedin 50 μl dH₂O and transferred to a 12×75 glass test tube containing 2.5ml of ice cold solution of 5% TCA and 10 mM Na pyrophosphate. The sampleis incubated on ice for 30 minutes. The sample is filtered through a 2.5cm wet (dH₂O) GFC membrane (S&S) on a vacuum filtration manifold. Thefilter is washed three times under vacuum with 5 ml ice cold 1% TCA, andonce with 5 ml 95% ethanol. The filter is dried and counted in ascintillation counter using scintillation fluid. The fmol of nucleotideincorporated is determined from the specific activity of radioactivetracer. The activity of extract is calculated based on the dNTPincorporated and is expressed as fmol dNTP/min./μl extract.

Telomerase Activity Assay

[0142] Bio-Tel Flash Plate Assay

[0143] An assay is provided for the detection and/or measurement oftelomerase activity by measuring the addition of TTAGGG telomericrepeats to a biotinylated telomerase substrate primer; a reactioncatalyzed by telomerase. The biotinylated products are captured instreptavidin-coated microtiter plates. An oligonucleotide probecomplementary to 3.5 telomere repeats labeled with [³³P] is used formeasuring telomerase products, as described below. Unbound probe isremoved by washing and the amount of probe annealing to the capturedtelomerase products is determined by scintillation counting.

[0144] Method:

[0145] I. 2′-Arabino-fluoro phosphoramidate oligonucleotides were storedas concentrated stocks and dissolved in PBS.

[0146] II. For testing, the 2′-arabino-fluoro phosphoramidateoligonucleotides were diluted to a 15×working stock in PBS and 2 μl wasdispensed into two wells of a 96-well microtiter dish (assayed induplicate).

[0147] III. Telomerase extract was diluted to a specific activity of0.04-0.09 fmol dNTP incorporated/min./μl in Telomerase Dilution Bufferand 18 μl added to each sample well to preincubate with compound for 30minutes at room temperature.

[0148] IV. The telomerase reaction was initiated by addition of 10 μlMaster Mix to the wells containing telomerase extract andoligonucleotide compound being tested. The plates were sealed andincubated at 37° C. for 90 min.

[0149] V. The reaction was stopped by the addition of 10 μl HCS.

[0150] VI. 25 μl of the reaction mixture was transferred to a 96-wellstreptavidin-coated FlashPlate (NEN) and incubated for 2 hours at roomtemperature with mild agitation.

[0151] VII. The wells were washed three times with 180 μl 2×SSC withoutany incubation.

[0152] VIII. The amount of probe annealed to biotinylated telomeraseproducts were detected in a scintillation counter. IX.

[0153] Buffers:

[0154] Telomerase Dilution Buffer

[0155] 50 mM Tris-acetate, pH 8.2

[0156] 1 mM DTT

[0157] 1 mM EGTA

[0158] 1 mM MgCl₂

[0159] 830 nM BSA

[0160] Master Mix (MM)

[0161] 50 mM Tris-acetate, pH 8.2

[0162] 1 mM DTT

[0163] 1 mM EGTA

[0164] 1 mM MgCl₂

[0165] 150 mM K acetate

[0166] 10 μM dATP

[0167] 20 μM dGTP

[0168] 120 μM dTTP

[0169] 100 nM biotinylated primer (5′-biotin-AATCCGTCGAGCAGAGTT-3′) [SEQID NO:14]

[0170] 5.4 nM labeled probe [5′-CCCTAACCCTAACCCTAACCC-(³³P)A₁₋₅₀-3′][SEQ ID NO:15]; specific activity approximately 10⁹ cpm/μg orhigher

[0171] Hybridization Capture Solution (HCS)

[0172] 12×SSC (1×=150 mM NaCl/30 mM Na₃Citrate)

[0173] 40 mM EDTA

[0174] 40 mM Tris-HCl, pH 7.0

[0175] Using the foregoing assay, IC₅₀ values for the inventive2′-arabino-fluoro phosphoramidate oligonucleotides represented by thesequences shown in Table 2 can be determined. TABLE 22′-ARABINO-FLUOROOLIGONUCLEOTIDES 1-4 AS TELOMERASE INHIBITORS INCOMPARISON WITH 2′-RIBOSE-FLUORO PHOSPHORAMIDATES: SEQ ID NOs5′-Oligonucleotide-3′^(a) SEQ ID NO:16dG-a(Tn^(f)Tn^(f)An^(f)Gn^(f)Gn^(f)Gn^(f)Tn^(f)Tn^(f)An^(f)Gn^(f)) SEQID NO:17dG-a(Tn^(f)Tn^(f)Gn^(f)An^(f)Gn^(f)Tn^(f)Gn^(f)Tn^(f)An^(f)Gn^(f)) SEQID NO:18dT-a(An^(f)Gn^(f)Gn^(f)Gn^(f)Tn^(f)Tn^(f)An^(f)Gn^(f)An^(f)Cn^(f)An^(f)An^(f)) SEQ ID NO:19dT-a(An^(f)Gn^(f)Gn^(f)Tn^(f)Gn^(f)Tn^(f)An^(f)An^(f)Gn^(f)Cn^(f)An^(f)An^(f)) SEQ ID NO:20dG-r(Tn^(f)Tn^(f)An^(f)Gn^(f)Gn^(f)Gn^(f)Tn^(f)Tn^(f)An^(f)Gn^(f)) SEQID NO:21dG-r(Tn^(f)Tn^(f)Gn^(f)An^(f)Gn^(f)Tn^(f)Gn^(f)Tn^(f)An^(f)Gn^(f)) SEQID NO:22dT-r(An^(f)Gn^(f)Gn^(f)Gn^(f)Tn^(f)Tn^(f)An^(f)Gn^(f)An^(f)Cn^(f)An^(f)An^(f)) SEQ ID NO:23dT-r(An^(f)Gn^(f)Gn^(f)Tn^(f)Gn^(f)Tn^(f)An^(f)An^(f)Gn^(f)Cn^(f)An^(f)An^(f))

[0176] Oligonucleotides SEQ ID NOs:17 and 19 are mismatch controls foroligonucleotides SEQ ID NOs:16 and 18, respectively, that are used tocompare the telomerase inhibiting power of 2′-arabino-fluorophosphoramidate oligonucleotides to corresponding 2′-ribose-fluorophoshoramidate analog oligonucleotides. Similarly, oligonucleotides SEQID NOs:21 and 23 are mismatch controls for the oligonucleotides SEQ IDNOs:20 and 22.

[0177] Based upon the thermal stability of the inventiveoligonucleotides (Table 1), it is anticipated that a telomeraseinhibition assay performed using 2′-arabino-fluoro phosphoramidatepolynucleotides SEQ ID NOs:16 and 18 of Table 2 will be more effectiveat inhibiting telomerase activity than their counterpart2′-ribose-fluorooligonucleotides SEQ ID NO:20 and 22. Thus, the2′-arabino-fluoro phosphoramidate oligonucleotides of the presentinvention are expected to be not only more active in the telomeraseinhibition assay as compared to their 2′-ribose-fluorooligonucleotidescounterparts, but are also more acid resistant than them as well (seeTable 1). This combination of characteristics imparts the inventive2′-arabino-fluoro phosphoramidate oligonucleotides with an importantadvantage compared to 2′-ribose-fluoro phosphoramidate polynucleotides.

EXAMPLE 7 Anti-Tumor Activity of 2′-Arabino-Fluoro PhosphoramidateOligonucleotides Ex Vivo Studies

[0178] a. Reduction of Telomere Length in Tumor Cells

[0179] Colonies of the tumor cell lines, such as the ovarian tumor celllines OVCAR-5, and SK-OV-3, and normal human cells used as a control(e.g., normal human BJ cells) are prepared using standard methods andmaterials. In one test, the colonies are prepared by seeding15-centimeter dishes with about 10⁶ cells in each dish. The dishes areincubated to allow the cell colonies to grow to about 80% confluence, atwhich time each of the colonies are divided into two groups. One groupis exposed to a subacute dose of a 2′-arabino-fluoro phosphoramidateoligonucleotide of the invention at a predetermined concentration (e.g.,between about 100 μM and about 20 μM) for a period of about 4-8 hoursafter plating following the split; the other group is exposed to acontrol (e.g., a phosphoramidate oligonucleotide complementary totelomerase RNA but having at least one base sequence that is mismatchedrelative to the sequence of telomerase RNA.

[0180] Each group is then allowed to continue to divide, and the groupsare split evenly again (near confluence). The same number of cells areseeded for continued growth. The test 2′-arabino-fluoro phosphoramidateoligonucleotide or control oligonucleotide is added every fourth day tothe samples at the same concentration delivered initially. Remainingcells are analyzed for telomere length. As the untested cell culturesnear confluence, the samples are split again as just described. Thissequence of cell doubling and splitting is continued for about 20 to 25doublings. Thus, a determination of telomere length as a function ofcell doublings is obtained.

[0181] Telomere length is determined by digesting the DNA of the cellsusing restriction enzymes specific for sequences other than therepetitive T₂ AG₃ sequence of human telomeres (TRF analysis). Thedigested DNA is separated by size using standard techniques of gelelectrophoresis to determine the lengths of the telomeric repeats, whichappear, after probing with a telomere DNA probe, on the gel as a smearof high-molecular weight DNA (approximately 2 Kb-15 Kb).

[0182] The results of the telomere length analysis are expected toindicate that the 2′-arabino-fluoro phosphoramidate oligonucleotides ofthe invention have no affect on the rate of decrease in telomere lengthfor control cells as a function of progressive cell doublings. Withrespect to the tumor cell lines, however, measurable decreases intelomere length are expected to be determined for tumor cells exposed tothe 2′-arabino-fluoro phosphoramidate oligonucleotides of the invention.Tumor cells exposed to the control oligonucleotides are expected tomaintain steady telomere lengths. Thus, the compounds of the inventionare expected to cause resumption of the normal loss of telomere lengthas a function of cell division in tumor cells.

[0183] In another experiment, HEK-293 cells are incubated with a2′-arabino-fluoro phosphoramidate oligonucleotide of the invention and acontrol oligonucleotide at concentrations between about 0.1 μM and about20 μM using the protocol just described. Cells are expected to entercrisis (i.e., the cessation of cell function) within several weeksfollowing administration of the test 2′-arabino-fluoro phosphoramidateoligonucleotides of the invention. In addition, TRF analysis of thecells using standard methodology is expected to show that the test2′-arabino-fluoro phosphoramidate oligonucleotides of the invention areeffective in reducing telomere length. In addition to the HEK-293 cellsdescribed above, this assay can be performed with anytelomerase-positive cell line, such as HeLa cells.

[0184] b. Specificity

[0185] Phosphoramidate 2′-arabino-fluoro oligonucleotides of theinvention are screened for activity (IC₅₀) against telomerase and otherenzymes known to have RNA components by performing hybridization testsor enzyme inhibition assays using standard techniques. Oligonucleotideshaving lower IC₅₀ values for telomerase as compared to the IC₅₀ valuestoward the other enzymes being screened are said to possess specificityfor telomerase.

[0186] C. Cytotoxicity

[0187] The XTT assay for cytotoxicity is performed using HeLa cells. Thecell lines used in the assay are exposed to a 2′-arabino-fluorophosphoramidate oligonucleotide of the invention for 72 hours atconcentrations ranging from about 1 μM to about 100 μM. During thisperiod, the optical density (OD) of the samples is determined for lightat 540 nanometers (nm). No significant cytotoxic effects are expected tobe observed at concentrations less than about 20 μM. It will beappreciated that other tumor cells lines such as the ovarian tumor celllines OVCAR-5 and SK-OV-3 can be used to determine cytotoxicity inaddition to control cell lines such as normal human BJ cells. Otherassays for cytotoxicity such as the MTT assay (see Berridge et al.,Biochemica 4:14-19, 1996) and the alamarBlue™ assay (U.S. Pat. No.5,501,959) can be used as well.

[0188] Preferably, to observe any telomerase inhibiting effects the2′-arabino-fluoro phosphoramidate oligonucleotides should beadministered at a concentration below the level of cytotoxicity.Nevertheless, since the effectiveness of many cancer chemotherapeuticsderives from their cytotoxic effects, it is within the scope of thepresent invention that the phosphoramidate oligonucleotides of thepresent invention be administered at any dose for which chemotherapeuticeffects are observed.

[0189] In vivo Studies

[0190] A human tumor xenograft model in which OVCAR-5 tumor cells aregrafted into nude mice can be constructed using standard techniques andmaterials. The mice are divided into two groups. One group is treatedintraperitoneally with a 2′-arabino-fluoro phosphoramidateoligonucleotides of the invention. The other group is treated with acontrol comprising a mixture of phosphate buffer solution (PBS) and anoligonucleotide complementary with telomerase RNA but has at least a onebase mismatch with the sequence of telomerase RNA. The average tumormass for mice in each group is determined periodically following thexenograft using standard methods and materials.

[0191] In the group treated with a 2′-arabino-fluoro phosphoramidateoligonucleotide of the invention, the average tumor mass is expected toincrease following the initial treatment for a period of time, afterwhich time the tumor mass is expected to stabilize and then begin todecline. Tumor masses in the control group are expected to increasethroughout the study. Thus, the 2′-arabino-fluoro phosphoramidateoligonucleotides of the invention are expected to lessen dramaticallythe rate of tumor growth and ultimately induce reduction in tumor sizeand elimination of the tumor.

[0192] Thus, the present invention provides novel 2′-arabino-fluorophosphoramidate oligonucleotides and methods for inhibiting telomeraseactivity and treating disease states in which telomerase activity hasdeleterious effects, especially cancer. The phosphoramidate2′-arabino-fluoro oligonucleotides of the invention provide a highlyselective and effective treatment for malignant cells that requiretelomerase activity to remain immortal; yet, without affectingnon-malignant cells.

[0193] While the preferred embodiment of the invention has beenillustrated and described, it will be appreciated that various changescan be made therein without departing from the spirit and scope of theinvention.

1 23 1 3 DNA Artificial Sequence contains 2 prime-deoxy and 2prime-arabino- fluoronucleosides 1 tuu 3 2 2 DNA Artificial Sequencecontains 2 prime-deoxy and 2 prime-arabino- fluoronucleosides 2 ut 2 310 DNA Artificial Sequence contains 2 prime-deoxy nucleosides 3uuuuuuuuut 10 4 10 DNA Artificial Sequence contains 2prime-ribo-fluoronucleosides 4 uuuuuuuuut 10 5 10 DNA ArtificialSequence contains 2 prime-arabino-nucleosides 5 uuuuuuuuut 10 6 10 DNAArtificial Sequence contains 2 prime-deoxy nucleosides 6 uuuuuuuuut 10 710 DNA Artificial Sequence contains 2 prime-deoxy and 2 prime-ribo-fluoronucleotides 7 tuuuuuuuuu 10 8 10 DNA Artificial Sequence contains2 prime-deoxy and 2 prime-arabino- fluoronucleosides 8 tuuuuuuuuu 10 9 9DNA Artificial Sequence contains 2 prime-deoxy nucleosides 9 cucucugcc 910 9 DNA Artificial Sequence contains 2 prime-arabino-fluoronucleosides10 cucucugcc 9 11 28 DNA Homo sapiens 11 tgtgtggtag acctgttacc agagggag28 12 26 DNA Homo sapiens 12 ctaaccctaa ctggtaacag gtctac 26 13 6 DNAHomo sapiens 13 ttaggg 6 14 18 DNA Homo sapiens 14 aatccgtcga gcagagtt18 15 21 DNA Homo sapiens 15 ccctaaccct aaccctaacc c 21 16 11 DNA Homosapiens 16 gttagggtta g 11 17 11 DNA Homo sapiens 17 gttgagtgta g 11 1813 DNA Homo sapiens 18 tagggttaga caa 13 19 13 DNA Homo sapiens 19taggtgtaag caa 13 20 11 DNA Homo sapiens 20 gttagggtta g 11 21 11 DNAHomo sapiens 21 gttgagtgta g 11 22 13 DNA Homo sapiens 22 tagggttaga caa13 23 13 DNA Homo sapiens 23 taggtgtaag caa 13

We claim:
 1. A polynucleotide comprising at least one2′-arabino-fluoronucleoside linked to at least one additional nucleosidesubunit by a N3′→P5′ phosphoramidate inter-subunit linkage.
 2. Apolynucleotide according to claim 1, wherein the N3′→P5′ phosphoramidatelinkage is defined by the formula: 3′-[—NH—P(—O)(OR)—O]—5′: wherein R isa positively charged counter ion or hydrogen.
 3. A polynucleotideaccording to claim 2, wherein all of the intersubunit linkages compriseN3′→P5′ phosphoramidate linkages, defined by the formula:

wherein B is a purine or pyrimidine or an analog thereof, R is apositively charged counter ion or hydrogen, R₁ is selected from thegroup consisting of hydroxyl, amino and hydrogen, and n is an integerbetween 1 and
 49. 4. A polynucleotide according to claim 1, whichadditionally comprises at least one second-type linkage selected fromthe group consisting of phosphodiester, phosphotriester,methylphosphonate, P3′→N5′ phosphoramidate, N3′→P5′ thiophosphoramidate,and phosphorothioate linkages.
 5. A polynucleotide according to claim 4,which comprises about 2 to 50 nucleoside subunits.
 6. A polynucleotideaccording to claim 1, wherein the polynucleotide additionally comprisesa reporter moiety.
 7. A polynucleotide according to claim 7, wherein thereporter moiety is selected from the group consisting of radioactivelabels, biotin labels, and fluorescent labels.
 8. A method ofsynthesizing a 2′-arabino-fluorooligonucleotide N3′→P5′ phosphoramidatecomprising the steps of: (a) providing a first 3′-amino protectednucleoside attached to a solid phase support; (b) deprotecting theprotected 3° amino group to form a free 3′ amino group; (c) reacting thefree 3′ amino group with a 3′-amino protected 2′-arabino-fluorophosphoramidite monomer to form an internucleoside N3′→P5′phosphoramidite linkage; and (d) oxidizing the internucleoside N3′→P5′linkage.
 9. A method of synthesizing a 2′-arabino-fluorooligonucleotideaccording to claim 8, further comprises the step of repeating aplurality of times the deprotecting, reacting and oxidizing steps.
 10. Amethod of synthesizing a 2′-arabino-fluorooligonucleotide according toclaim 8, wherein the deprotecting, reacting and oxidizing steps arerepeated from 2 to 4 times.
 11. A method of synthesizing a2′-arabino-fluorooligonucleotide according to claim 8, wherein the3′-amino protected 2′-arabino-fluoro phosphoramidite monomer comprises a3′-(monomethoxytrityl)-amino-5′-O-(cyanoethyl-N,N′-diisopropylamino)-phosphoramiditenucleoside.
 12. A method of synthesizing a2′-arabino-fluorooligonucleotide according to claim 11, wherein the3′-(monomethoxytrityl)-amino-5′-O-(cyanoethyl-N,N′-diisopropylamino)-phosphoramiditenucleoside comprises a base selected from the group consisting ofadenine, guanine, 2,6-diaminopurine, uracil, cytosine and thymidine. 13.A method of synthesizing a 2′-arabino-fluorooligonucleotide according toclaim 12, further comprising the step of capping the free 3′-aminogroups that fail to react with the 3′-amino protected 2′-arabino-fluorophosphoramidite monomer.
 14. A compound of the formula:

wherein: B is a purine or pyrimidine or an analog thereof; R₂ is H,lower alkyl, PO₃, or PN(R₄)₂OR₅ wherein R₄ is dialkyl, and R₅ iscyano-lower alkyl; and R₃ is hydrogen or substituted or unsubstitutedtrityl.
 15. A compound according to claim 14, wherein R₂ is PN(R₄)₂OR₅wherein R₄ is diisopropyl, R₅ is β-cyanoethyl and R₃ ismonomethoxytrityl.
 16. A compound according to claim 14, wherein theconstituent B is exocyclic amino protected.
 17. A compound according toclaim 14, wherein B is guanine and the N² amino group of guanine isprotected with an isobutyl group, B is 2,6-diaminopurine and theexocylic amine groups of 2,6-diaminopurine are protected with aphenoxyacetyl group, or B is cytosine and the N4 amino group of cytosineis protected with a benzoyl group.
 18. A method of hybridizingpolynucleotide to a DNA or RNA target comprising contacting apolynucleotide according to claim 1 with the target under conditionsthat allow formation of a hybridization complex between thepolynucleotide and the target.
 19. A method according to claim 18,wherein the polynucleotide carries a reporter moiety.
 20. A methodaccording to claim 19, wherein the reporter moiety is selected from thegroup consisting of radioactive labels, biotin labels, and fluorescentlabels.
 21. A method for detecting a target nucleic acid in a sample,comprising: a) preparing a reaction mixture comprising the sample and apolynucleotide according to claim 1 capable of hybridizing specificallywith the target nucleic acid; b) detecting hybrids formed in thereaction mixture; and c) correlating any hybrids formed with thepresence of the target nucleic acid sequence in the sample.
 22. A methodfor isolating a target nucleic acid sequence from a sample, comprising:a) combining the sample and a polynucleotide according to claim 1capable of hybridizing specifically with the target sequence; and b)recovering the target nucleic acid from hybrids formed with thepolynucleotide.
 23. A method for inhibiting function of an RNA in acell, comprising contacting the cell with a polynucleotide according toclaim 1 that can specifically hybridize with the RNA.
 24. A methodaccording to claim 23, which is a method for inhibiting translation ofan mRNA, wherein the polynucleotide comprises a sequence containing atleast 10 bases complementary to a sequence contained in the mRNA.
 25. Amethod according to claim 23, which is a method for inhibitingtelomerase enzyme in a cell, wherein the polynucleotide comprises asequence complementary to telomerase RNA component.
 26. A method forinhibiting activity of a telomerase enzyme in a cell comprisingcontacting the cell with an effective amount of a polynucleotideaccording to claim 1, wherein the polynucleotide comprises a sequencecomplementary to telomerase RNA component.
 27. A kit for determining orisolating a nucleic acid containing a specific sequence in a sample,comprising a polynucleotide according to claim 1 that can hybridize tothe specific sequence, and written indications for using thepolynucleotide for determining or isolating the nucleic acid.
 28. Use ofa polynucleotide according to any of claims 1-9, in the preparation of amedicament for treatment of the human or animal body.
 29. Use of apolynucleotide according to any of claims 1-9, in the preparation of amedicament for treatment of a viral infection.
 30. Use of apolynucleotide according to any of claims 1-9, in the preparation of amedicament for treatment of cancer.
 31. The use according to any ofclaims 28-30, wherein the polynucleotide can hybridize with telomeraseRNA component.